Micromirs

ABSTRACT

The present invention relates to very short heavily modified oligonucleotides which target and inhibit microRNAs in vivo, and their use in medicaments and pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. Nonprovisional application Ser.No. 14/527,115, filed Oct. 29, 2014, (U.S. Pat. No. 9,790,493) which isa Continuation of U.S. Nonprovisional application Ser. No. 12/681,587,filed Aug. 11, 2010, (U.S. Pat. No. 8,906,871) which is a National Stageof Application No. PCT/DK2008/000344, filed Oct. 3, 2008, which claimsthe benefit of U.S. Provisional Application Nos. 61/028,062, filed Feb.12, 2008, 60/979,217, filed Oct. 11, 2007, and 60/977,497, filed Oct. 4,2007; and European Patent Application No. 08104780, filed Jul. 17, 2008,all of which are incorporated herein by reference in their entireties.Furthermore we reference and incorporate by reference W02007/112754 andW02007/112753 which are earlier applications from the same applicants.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:2763_013006_sequence_listing_ST25.txt; Size: 598.545 bytes; and Date ofCreation: Oct. 19, 2014) is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to very short oligonucleotides whichtarget and inhibit microRNAs in vivo, and their use in medicaments andpharmaceutical compositions.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are an abundant class of short endogenous RNAs thatact as post-transcriptional regulators of gene expression bybase-pairing with their target mRNAs. They are processed from longer (ca70-80 nt) hairpin-like precursors termed pre-miRNAs by the RNAse IIIenzyme Dicer. MicroRNAs assemble in ribonucleoprotein complexes termedmiRNPs and recognize their target sites by antisense complementaritythereby mediating down-regulation of their target genes. Near-perfect orperfect complementarity between the miRNA and its target site results intarget mRNA cleavage, whereas limited complementarity between themicroRNA and the target site results in translational inhibition of thetarget gene.

A summary of the role of microRNAs in human diseases, and the inhibitionof microRNAs using single stranded oligonucleotides is provided byWO2007/112754 and WO2007/112753, which are both hereby incorporated byreference in its entirety. WO2008046911, hereby incorporated byreference, provides microRNA sequences which are associated with cancer.Numerous microRNAs have been associated with disease phenotypes and itis therefore desirable to provide substances capable of modulating theavailability of microRNAs in vivo. WO2007/112754 and WO2007/112753disclose short single stranded oligonucleotides which are considered toform a strong duplex with their target miRNA. SEQ ID NOs 1-45 areexamples of anti microRNA oligonucleotides as disclosed in WO2007/112754and WO2007/112753.

RELATED APPLICATIONS

This application claims priority from four applications: U.S. 60/977,497filed 4 Oct. 2007, U.S. 60/979,217 filed 11 Oct. 2007, U.S. 61/028,062,filed 12 Feb. 2008, and EP08104780, filed 17 Jul. 2008, all of which arehereby incorporated by reference. Furthermore we reference andincorporate by reference WO2007/112754 and WO2007/112753 which areearlier applications from the same applicants.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that the use of veryshort oligonucleotides which target microRNAs and which have a highproportion of nucleotide analogue nucleotides, such as LNA nucleotides,are highly effective in alleviating the repression of RNAs, such as anmRNA, by the targeted microRNAs in vivo.

The present invention provides an oligomer a contiguous sequence of 7,8, 9 or 10 nucleotide units in length, for use in reducing the effectiveamount of a microRNA target in a cell or an organism, wherein at least70%, such as at least 80% of the nucleotide units of the oligomer areselected from the group consisting of LNA units and 2′ substitutednucleotide analogues.

The present invention provides an oligomer a contiguous sequence of 7,8, 9 or 10 nucleotide units in length, for use in reducing the effectiveamount of a microRNA target in a cell or an organism, wherein at least70% of the nucleotide units of the oligomer are selected from the groupconsisting of LNA units and 2′ substituted nucleotide analogues, andwherein at least 50%, such as at least 60%, such as at least 70% of thenucleotide units of the oligomer are LNA units.

The invention provides oligomers of between 7-10 nucleotides in lengthwhich comprises a contiguous nucleotide sequence of a total of between7-10 nucleotides, such as 7, 8, 9, nucleotide units, wherein at least50% of the nucleotide units of the oligomer are nucleotide analogues.

The invention further provides for an oligomer of between 7-10nucleotides in length which comprises a contiguous nucleotide sequenceof a total of between 7-10 nucleotides, such as 7, 8, 9, or 10,nucleotide units, wherein the nucleotide sequence is complementary to acorresponding nucleotide sequence found in mammalian or viral microRNA,and wherein at least 50% of the nucleotide units of the oligomer arenucleotide analogues.

The present invention provides olgiomers according to the invention as amedicament.

The present invention provides pharmaceutical compositions comprisingthe oligomer of the invention and a pharmaceutically acceptable diluent,carrier, salt or adjuvant.

The invention provides for a conjugate comprising an oligomer accordingto the invention, conjugated to at least one non-nucleotide orpolynucleotide entity, such as a sterol, such as cholesterol.

The invention provides for the use of an oligomer or a conjugateaccording to the invention, for the manufacture of a medicament for thetreatment of a disease or medical disorder associated with the presenceor over-expression of a microRNA, such as one or more of the microRNAsreferred to herein.

The invention provides for the treatment of a disease or medicaldisorder associated with the presence or overexpression of the microRNA,comprising the step of administering a composition (such as thepharmaceutical composition) comprising an oligomer or conjugateaccording to the invention to a patient suffering from or likely tosuffer from said disease or medical disorder.

The invention provides for a method for reducing the effective amount ofa microRNA target in a cell or an organism, comprising administering theoligomer of the invention, or a composition (such as a pharmaceuticalcomposition) comprising the oligomer or conjugate according to theinvention to the cell or organism.

The invention provides for a method for reducing the effective amount ofa microRNA target in a cell or an organism, comprising administering theoligomer or conjugate or pharmaceutical composition according to theinvention to the cell or organism.

The invention provides for a method for de-repression of a target mRNA(or one ore mor RNAs) in a cell or an organism, comprising administeringan oligomer or conjugate according to the invention, or a compositioncomprising said oligomer or conjugate, to said cell or organism.

The invention provides for the use of an oligomer or a conjugateaccording to the invention, for inhibiting the mircoRNA in a cell whichcomprises said microRNA, such as a human cell. The use may be in vivo orin vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic presentation of the miR-21, miR-155 and miR-122 8-merLNA-antimiRs, indicating the targeting positions with the fullyLNA-modified and phosphorothiolated LNA-antimiR. Preferred hybridisationpositions for 7mer, 8mer, 9mer and 10 mer LNA oligonucleotides on themature microRNA are also indicated.

FIG. 2. Assessment of miR-21 antagonism by SEQ ID #3205 and SEQ ID #3204LNA-antimiRs in MCF-7 cells using a luciferase sensor assay. MCF-7 cellswere co-transfected with luciferase sensor plasmids containing a perfectmatch target site for miR-21 or a mismatch target site (·mm2) andLNA-antimiRs at different concentrations. After 24 hours, cells wereharvested and luciferase activity measured. Shown are the mean ofrenilla/firefly ratios for three separate experiments (bars=s.e.m), wereall have been normalized against 0 nM psiCHECK2 (=control).

FIG. 3. Assessment of miR-21 antagonism by SEQ ID #3205 and SEQ ID #3204LNA-antimiRs in HeLa cells using a luciferase sensor assay. HeLa cellswere co-transfected with luciferase sensor plasmids containing a perfectmatch target site for miR-21 (mir-21) or a mismatch target site (mm2)and LNA-antimiRs at different concentrations. After 24 hours, cells wereharvested and luciferase activity measured. Shown are the mean ofrenilla/firefly ratios for three separate experiments (bars=s.e.m), wereall have been normalized against 0 nM psiCHECK2 (=control).

FIG. 4. Assessment of miR-155 antagonism by SEQ ID #3206 and SEQ ID#3207 LNA-antimiRs in LPS-treated mouse RAW cells using a luciferasesensor assay. RAW cells were co-transfected with miR-155 and thedifferent LNA-antimiRs at different concentrations. After 24 hours,cells were harvested and luciferase activity measured. Shown are themean of renilla/firefly, were all have been normalized against 0 nMpsiCHECK2.

FIG. 5. Assessment of miR-122 antagonism by SEQ ID #3208 and SEQ ID #4LNA-antimiRs in HuH-7 cells using a luciferase sensor assay. HuH-7 cellswere co-transfected with a miR-122 luciferase sensor containing aperfect match miR-122 target site and the different LNA-antimiRs atdifferent concentrations. After 24 hours, cells were harvested andluciferase activity measured. Shown are the mean of renilla/fireflyratios for three separate experiments (bars=s.e.m), where all have beennormalized against 0 nM psiCHECK2 (=control).

FIG. 6. Schematic presentation of the miR-21 luciferase reporterconstructs.

FIG. 7. Assessment of miR-21 antagonism by an 8-mer LNA-antimiR (SEQ ID#3205) versus a 15-mer LNA-antimiR (SEQ ID #3204) in PC3 cells using aluciferase reporter assay. PC3 cells were co-transfected with luciferasereporter plasmids containing a perfect match target site for miR-21 or amismatch target site and LNA-antimiRs at different concentrations. After24 hours, cells were harvested and luciferase activity measured. Shownare the mean values (bars=s.e.m) of three independent experiments wherethe renilla/firefly ratios have been normalized against 0 nM emptyvector without target site (=control). Shown is also a schematicpresentation of the miR-21 sequence and the design and position of theLNA-antimiRs. LNA nucleotides are indicated by ovals, and DNA residuesare indicated by bars.

FIG. 8. Specificity assessment of miR-21 antagonism by an 8-merLNA-antimiR in HeLa cells using a luciferase reporter assay. HeLa cellswere co-transfected with luciferase reporter plasmids containing aperfect match or a mismatched target site for miR-21 and LNA-antimiRs(SEQ ID #3205) or an 8-mer LNA mismatch control oligo (SEQ ID #3218) atdifferent concentrations. After 24 hours, cells were harvested andluciferase activity was measured. Shown are the mean values (bars=s.e.m)for three independent experiments where the Renilla/firefly ratios havebeen normalized against 0 nM empty vector without target site(=control). Shown is also a schematic presentation of the miR-21sequence and the design and position of the LNA-antimiRs. Mismatches areindicated by filled ovals.

FIG. 9. Assessment of the shortest possible length of a fullyLNA-modified LNA-antimiR that mediates effective antagonism of miR-21.HeLa cells were co-transfected with luciferase reporter plasmidscontaining a perfect match or a mismatch target site for miR-21 and theLNA-antimiRs at different concentrations (SEQ ID #3209=6-mer and SEQ ID#3210=7-mer). After 24 hours, cells were harvested and luciferaseactivity measured. Shown are the mean values (bars=s.e.m) for threeindependent experiments where the renilla/firefly ratios have beennormalized against 0 nM empty vector without target site (=control).Shown is also a schematic presentation of the miR-21 sequence and thedesign and position of the LNA-antimiRs.

FIG. 10. Length assessment of fully LNA-substituted LNA-antimiRsantagonizing miR-21. HeLa cells were co-transfected with luciferasereporter plasmids containing a perfect match or a mismatch target sitefor miR-21 and LNA-antimiRs at different concentrations (SEQ ID#3211=9-mer, SEQ ID #3212=10-mer, SEQ ID #3213=12-mer and SEQ ID#3214=14-mer). After 24 hours, cells were harvested and luciferaseactivity measured. Shown are the mean values (bars=s.e.m) for threeindependent experiments where the renilla/firefly ratios have beennormalized against 0 nM empty vector without target site (=control).Shown is also a schematic presentation of the miR-21 sequence and thedesign and position of the LNA-antimiRs.

FIG. 11. Determination of the most optimal position for an 8-merLNA-antimiR within the miR target recognition sequence. HeLa cells wereco-transfected with luciferase reporter plasmids containing a perfectmatch or a mismatch target site for miR-21 and the LNA-antimiRs atdifferent concentrations. After 24 hours, cells were harvested andluciferase activity measured. Shown are the mean values (bars=s.e.m) forthree independent experiments where the renilla/firefly ratios have beennormalized against 0 nM empty vector without target site (=control).Shown is also a schematic presentation of the miR-21 sequence and thedesign and position of the LNA-antimiRs.

FIG. 12. Validation of interaction of the Pdcd4-3′-UTR and miR-21 by the8-mer SEQ ID #3205 LNA-antimiR. HeLa cells were co-transfected with aluciferase reporter plasmid containing part of the 3′UTR of Pdcd4 geneand LNA-antimiRs at different concentrations (SEQ ID #3205=8 mer,perfect match; SEQ ID #3218=8 mer, mismatch; SEQ ID #3204=15 mer,LNA/DNA mix; SEQ ID #3220=15 mer, gapmer). After 24 hours, cells wereharvested and luciferase activity measured. Shown are renilla/fireflyratios that have been normalized against 0 nM. Shown is also a schematicpresentation of the miR-21 sequence and the design and position of theLNA-antimiRs.

FIG. 13. Comparison of an 8-mer LNA-antimiR (SEQ ID #3207) with a 15-merLNA-antimiR (SEQ ID #3206) in antagonizing miR-155 in mouse RAW cells.Mouse RAW cells were co-transfected with luciferase reporter plasmidscontaining a perfect match for miR-155 and the different LNA-antimiRs atdifferent concentrations. After 24 hours, cells were harvested andluciferase activity measured. Shown are the mean values (bars=s.e.m) ofthree independent experiments where the renilla/firefly ratios have beennormalized against 0 nM empty vector without miR-155 target site(=control). Shown is also a schematic presentation of the miR-155sequence and the design and position of the LNA-antimiRs.

FIG. 14. Assessment of c/EBPOAssessment of c/EBPer LNA-antimiR (SEQ ID#3207) with a 15-mer LNA-antimiR (SEQ ID #3206) in antagonizing miR-155in mouse RAW cells. Mouse RAW cells were co-transfected with luciferasereporter plasmids containing a perfect match for miR-155 and thedifflter 20 hours, cells were harvested and western blot analysis ofprotein extracts from RAW cells was performed. The different isoforms ofc/EBPβ are indicated, and the ratios calculated on c/EBPβ LIP andbeta-tubulin are shown below.

FIG. 15. Antagonism of miR-106b by a fully LNA-modified 8-mer (SEQ ID#3221) LNA-antimiR or by a 15-mer mixmer (SEQ ID #3228) antimiR. HeLacells were co-transfected with luciferase reporter plasmids containing aperfect match for miR-106b and the different LNA-antimiRs at differentconcentrations. After 24 hours, cells were harvested and luciferaseactivity measured. Shown are the mean values of four replicates wherethe renilla/firefly ratios have been normalized against 0 nM emptyvector without miRNA target site (=control). Shown is also a schematicpresentation of the miR-106b sequence and the design and position of theLNA-antimiRs.

FIG. 16. Antagonism of miR-19b by a fully LNA-modified 8-mer (SEQ ID#3222) LNA-antimiR and a 15-mer (SEQ ID #3229) mixmer antimiR. HeLacells were co-transfected with luciferase reporter plasmids containing aperfect match for miR-19a and the two LNA-antimiRs at differentconcentrations. After 24 hours, cells were harvested and luciferaseactivity measured. Shown are the mean values of four replicateexperiments, where the renilla/firefly ratios have been normalizedagainst 0 nM empty vector without a miR-19a target site (=control).Shown is also a schematic presentation of the miR-19a sequence and thedesign and position of the LNA-antimiRs.

FIG. 17. Schematic presentation showing the mature human miR-221 (SEQ IDNO: 432) and miR-222 (SEQ ID NO: 3788) sequences. Shown in the square isthe seed sequence (7-mer) that is conserved in both miRNA sequences.

FIG. 18. Targeting of a microRNA family using short, fullyLNA-substituted LNA-antimiR. PC3 cells were co-transfected withluciferase reporter plasmids for miR-221 and miR-222 separately ortogether and with the different LNA-antimiRs at varying concentrations.When co-transfecting with the LNA-antimiRs (15-mers) SEQ ID #3223(against miR-221) and SEQ ID #3224 (against miR-222), the totalconcentration was 2 nM (1 nM each), while transfecting the cells withSEQ ID #3225 (7-mer) the concentrations were 0, 1, 5, 10 or 25 nM. After24 hours, cells were harvested and luciferase activity measured. Shownare the mean values (bars=s.e.m) of three independent experiments wherethe renilla/firefly ratios have been normalized against 0 nM emptyvector without a miRNA target site (=control). Shown is also a schematicpresentation of the miR-221/222 sequence and the design and position ofthe LNA-antimiRs.

FIG. 19. Assessment of p27 protein levels as a functional readout forantagonism of the miR-221/222 family by the 7-mer SEQ ID #3225LNA-antimiR. PC3 cells were transfected with the 7-mer LNA-antimiR SEQID #3225 targeting both miR-221 and miR-222 at varying concentrations.After 24 hours, cells were harvested and protein levels were measured ona western blot. Shown are the ratios of p27/tubulin.

FIG. 20. Assessment of miR-21 antagonism by an 8-mer LNA-antimiR (SEQ ID#3205) versus a 15-mer LNA-antimiR (SEQ ID #3204) and an 8-mer with 2mismatches (SEQ ID #3218) in HepG2 cells using a luciferase reporterassay.

HepG2 cells were co-transfected with luciferase reporter plasmidcontaining a perfect match target site for miR-21 and LNA-antimiRs atdifferent concentrations. After 24 hours, cells were harvested andluciferase activity measured. Shown are the mean values (bars=s.e.m) ofthree independent experiments where the renilla/firefly ratios have beennormalized against 0 nM empty vector without target site (=control).Shown is also a schematic presentation of the miR-21 sequence and thedesign and position of the LNA-antimiRs.

FIG. 21. Validation of interaction of the Pdcd4 3′UTR and miR-21 by the8-mer SEQ ID #3205 LNA-antimiR versus the 15-mer (SEQ ID #3204) and an8-mer with two mismatches (SEQ ID #3218).

Huh-7 cells were co-transfected with a luciferase reporter plasmidcontaining part of the 3′UTR of Pdcd4 gene, pre-miR-21 (10 nM) andLNA-antimiRs at different concentrations. After 24 hours, cells wereharvested and luciferase activity measured. Shown are the mean values(bars=s.e.m) of three independent experiments where the renilla/fireflyratios have been normalized against 0 nM empty vector without targetsite (=control). Shown is also a schematic presentation of the miR-21sequence and the design and position of the LNA-antimiRs.

FIG. 22. Antagonism of miR-21 by SEQ ID #3205 leads to increased levelsof Pdcd4 protein levels.

HeLa cells were transfected with 5 nM LNA-antimiR SEQ ID #3205 (perfectmatch), or SEQ ID #3219 LNA scrambled (8mer) or SEQ ID #3218 (8-mermismatch). Cells were harvested after 24 hours and subjected to Westernblot with Pdcd4 antibody.

FIG. 23. ALT and AST levels in mice treated with SEQ ID #3205 (perfectmatch) or SEQ ID #3218 (mismatch control). Mice were sacrificed after 14days and after receiving 25 mg/kg every other day.

FIG. 24. Assessment of PU.1 protein levels as a functional readout formiR-155 antagonism by short LNA-antimiR (SEQ ID #3207).

THP-1 cells were co-transfected with pre-miR-155 (5 nmol) and differentLNA oligonucleotides (5 nM) and 100 ng/ml LPS was added. After 24 hours,cells were harvested and western blot analysis of protein extracts fromthe THP-1 cells was performed. PU.1 and tubulin are indicated.

FIG. 25. Assessment of p27 protein levels as a functional readout forantagonism of the miR-221/222 family by the 7-mer SEQ ID #3225LNA-antimiR.

PC3 cells were transfected with the 7-mer LNA-antimiR SEQ ID #3225targeting both miR-221 and miR-222 and a LNA scrambled control at 5 and25 nM. After 24 hours, cells were harvested and protein levels weremeasured on a western blot. Shown are the ratios of p27/tubulin.

FIG. 26. Knock-down of miR-221/222 by the 7-mer SEQ ID #3225 (perfectmatch) LNA-antimiR reduces colony formation in soft agar in PC3 cells.

PC3 cells were transfected with 25 nM of the 7-mer LNA-antimiR SEQ ID#3225 targeting both miR-221 and miR-222 or a 7-mer scrambled control((SEQ ID #3231). After 24 hours, cells were harvested and seeded on softagar. After 12 days, colonies were counted. One experiment has been donein triplicate.

FIG. 27. Overview of the human let-7 family, and of tested antagonists.(upper) The sequences represent the mature miRNA for each member and thebox depicts nucleotides 2-16, the positions typically antagonized byLNA-antimiRs. Columns to the right show the number of nucleotidedifferences compared to let-7a, within the seed (S:position 2-8),extended seed (ES; position 2-9), and the remaining sequence typicallytargeted by LNA-antimiRs (NE; position 9-16), respectively. Nucleotideswith inverted colors are altered compared to let-7a. (lower) Summary oftested antagonists against the let-7 family, including information ondesign, length and perfectly complementary targets. All compounds arefully phoshorothiolated. (SEQ ID Nos: 24, 33, 34, 35, 36, 37, 25, 29, 31and 32)

FIG. 28. Assessment of let-7 antagonism by six different LNA-antimiRs inHuh-7 cells using a luciferase sensor assay.

Huh-7 cells were co-transfected with luciferase sensor plasmidscontaining a partial HMGA2 3′UTR (with four let-7 binding sites), withor without let-7a precursor (grey and black bars, respectively), andwith 6 different LNA-antimiRs at increasing concentrations. After 24hours, cells were harvested and luciferase activity measured. Shown arethe mean of renilla/firefly ratios for duplicate measurements andstandard deviations for each assay. Within each LNA-antimiR group allratios have been normalized to the average of wells containing no let-7aprecursor (black bars).

FIG. 29. Luciferase results from Huh-7 cells transfected with the HMGA23′UTR sensor plasmid, LNA-antimiRs SEQ ID #3226 (left) and SEQ ID #3227(right), and pre-miRs for let-7a (A), let-7d (B), let-7e (C), and let-7i(D). Grey bars indicate the target de-repression after pre-misinclusion, whereas black control bars represent the equivalent levelwithout pre-miR addition. Each ratio is based on quadruplicatemeasurements and have been normalized against the average of wellscontaining no precursor (black bars) within each treatment group.

FIG. 30. Luciferase results from HeLa cells transfected with the HMGA23′UTR sensor plasmid or control vector, and the LNA-antimiR SEQ ID #3227at various concentrations. Each ratio is based on quadruplicatemeasurements normalized against untreated (0 nM) empty control vector(psi-CHECK-2; grey bars).

FIG. 31. Assessment of miR-21 antagonism by 8mer (#3205) in HCT116 cellsusing a luciferase sensor assay. HCT116 cells were co-transfected withluciferase sensor plasmids containing a perfect match target site formiR-21 (grey bars) and LNA-antimiR and control oigonucleotides atdifferent concentrations. After 24 hours, cells were harvested andluciferase activity measured. Shown is one typical example of two wherethe renilla/firefly ratios have been normalized against 0 nM emptyvector (=black bars).

FIG. 32. Silencing of miR-21 by the 8-mer #3205 LNA-antimiR reducescolony formation in soft agar in PC3 cells. PC3 cells were transfectedwith 25 nM of the 8-mer LNA-antimiR #3205 targeting miR-21. After 24hours, cells were harvested and seeded on soft agar. After 12 days,colonies were counted. Shown is the mean of three separate experiments,each performed in triplicate, and normalised against 0 nM control (i.e.transfection but with no LNA). p=0.01898 for #3205.

FIG. 33. Knock-down of miR-21 by the 8-mer #3205 LNA-antimiR reducescolony formation in soft agar in HepG2 cells. HepG2 cells weretransfected with 25 nM of the 8-mer LNA-antimiR #3205 targeting miR-21.After 24 hours, cells were harvested and seeded on soft agar. After 17days, colonies were counted. Shown is the mean of three replicates fromone experiment (bars=SEM).

FIG. 34A. Wound closure in the invasive human prostate cell line PC3after treatment with #3205. PC3 cells were transfected at day 3 withLNA-antimiR and control oligonucleotides at 25 nM, #3205 (8mer, perfectmatch) and #3219 (8mer, mismatch) and the following day a scratch wasmade. Pictures were taken after 24 hours in order to monitor themigration. FIG. 34B The area in each timepoint has been measured withthe software program Image J and normalized against respective 0 htime-point.

FIG. 35. Length assessment of fully LNA-substituted LNA-antimiRsantagonizing miR-155. RAW cells were co-transfected with luciferasereporter plasmids containing a perfect match target site for miR-155 andwith LNA-antimiR oligonucleotides at different concentrations. After 24hours, cells were harvested and luciferase activity measured. Shown arethe mean values (bars=s.e.m) for three independent experiments where therenilla/firefly ratios have been normalized against 0 nM empty vectorwithout target site (=mock). Shown is also a schematic presentation ofthe miR sequence and the design and position of the LNA-antimiRs.

FIG. 36A. Binding of 5′-FAM labeled LNA-antimiR-21 (#3205) to mouseplasma protein. The drawing shows % unbound LNA-antimiR-21 compound as afunction of oligonucleotide concentration in mouse plasma. FIG. 36BConcentration of unbound LNA-antimiR-21 compound #3205 as a function of#3205 concentration in mouse plasma.

FIGS. 37A and B. Quantification Ras protein levels by Western blotanalysis.

-   -   A. Gel image showing Ras and Tubulin (internal standard) protein        in treated (anti-let-7; 8-mer) vs. untreated (saline) lung and        kidney samples. B. Quantifications of Ras protein levels in the        lung and kidney, respectively, of LNA-antimiR-treated mice        (black bars), normalized against equivalent saline controls        (grey bars), using tubulin as equal-loading control.    -   B. Silencing of miR-21 by #3205 leads to increased levels of        Pdcd4 protein levels in vivo. Mice were injected with saline or        25 mg/kg LNA-antimiR (#3205) over 14 days every other day, with        a total of 5 doses. Mice were sacrificed and protein was        isolated from kidney and subjected to Western blot analysis with        Pdcd4 antibody. A. Gel image showing Pdcd4 and Gapdh (internal        standard) protein in treated (antimiR-21; 8-mer) vs. untreated        (saline) kidney samples (M1, mouse 1; M2, mouse 2). B.        Quantification of Pdcd4 protein levels in kidneys of        LNA-antimiR-treated mice (dark grey bars), normalized against        the average of equivalent saline controls (light grey bars),        using Gapdh as loading control.

FIG. 38A. Silencing of miR-21 by Compound 3205 (SEQ. ID NO: 2) leads toincreased levels of Pdcd4 protein levels in vivo. Mice were injectedwith saline or 25 mg/kg LNA-antimiR Compound 3205 (SEQ ID NO: 2) over 14days every other day, with a total of 5 doses. Mice were sacrificed andprotein was isolated from kidney and subjected to Western blot analysiswith Pdcd4 antibody. Gel image showing Pdcd4 and Gapdh (internalstandard) protein in treated (antimiR-21; 8-mer) vs. untreated (saline)kidney samples (M1, mouse 1; M2, mouse 2).

FIG. 38B. Quantification of Pdcd4 protein levels in kidneys ofLNA-antimiR-treated mice (dark grey bars), normalized against theaverage of equivalent saline controls (light grey bars), using Gapdh asloading control.

DETAILED DESCRIPTION OF THE INVENTION

Short oligonucleotides which incorporate LNA are known from the in vitroreagents area, (see for example WO2005/098029 and WO 2006/069584).However the molecules designed for diagnostic or reagent use are verydifferent in design than those for in vivo or pharmaceutical use. Forexample, the terminal nucleotides of the reagent oligos are typicallynot LNA, but DNA, and the internucleoside linkages are typically otherthan phosphorothioate, the preferred linkage for use in theoligonucleotides of the present invention. The invention thereforeprovides for a novel class of oligonucleotides (referred to herein asoligomers) per se.

The following embodiments refer to certain embodiments of the oligomerof the invention, which may be used in a pharmaceutical composition.Aspects which refer to the oligomer may also refer to the contiguousnucleotide sequence, and vice versa.

The Oligomer

The oligomer of the invention is a single stranded oligonucleotide whichcomprises nucleotide analogues, such as LNA, which form part of, or theentire contiguous nucleotide sequence of the oligonucleotide. Thenucleotide sequence of the oligomer consists of a contiguous nucleotidesequence.

The term “oligonucleotide” (or simply “oligo”), which is usedinterchangeably with the term “oligomer” refers, in the context of thepresent invention, to a molecule formed by covalent linkage of two ormore nucleotides. When used in the context of the oligonucleotide of theinvention (also referred to the single stranded oligonucleotide), theterm “oligonucleotide” may have, in one embodiment, for example havebetween 7-10 nucleotides, such as in individual embodiments, 7, 8, 9, or10.

The term ‘nucleotide’ refers to nucleotides, such as DNA and RNA, andnucleotide analogues. It should be recognised that, in some aspects, theterm nucleobase may also be used to refer to a nucleotide which may beeither naturally occurring or non-naturally occurring—in this respectthe term nucleobase and nucleotide may be used interchangeably herein.

In some embodiments, the contiguous nucleotide sequence consists of 7nucleotide analogues. In some embodiments, the contiguous nucleotidesequence consists of 8 nucleotide analogues. In some embodiments, thecontiguous nucleotide sequence consists of 9 nucleotide analogues.

In one embodiment at least about 50% of the nucleotides of the oligomerare nucleotide analogues, such as at least about 55%, such as at leastabout 60%, or at least about 65% or at least about 70%, such as at leastabout 75%, such as at least about 80%, such as at least about 85%, suchas at least about 90%, such as at least about 95% or such as 100%. Itwill also be apparent that the oligonucleotide may comprise of anucleotide sequence which consists of only nucleotide analogues.Suitably, the oligomer may comprise at least one LNA monomer, such as 2,3, 4, 5, 6, 7, 8, 9 or 10 LNA monomers. As described below, thecontiguous nucleotide sequence may consist only of LNA units (includinglinkage groups, such as phosphorothioate linkages), or may consist ofLNA and DNA units, or LNA and other nucleotide analogues. In someembodiments, the contiguous nucleotide sequence comprises either one ortwo DNA nucleotides, the remainder of the nucleotides being nucleotideanalogues, such as LNA unit.

In some embodiments, the contiguous nucleotide sequence consists of 6nucleotide analogues and a single DNA nucleotide. In some embodiments,the contiguous nucleotide consists of 7 nucleotide analogues and asingle DNA nucleotide. In some embodiments, the contiguous nucleotidesequence consists of 8 nucleotide analogues and a single DNA nucleotide.In some embodiments, the contiguous nucleotide sequence consists of 9nucleotide analogues and a single DNA nucleotide. In some embodiments,the contiguous nucleotide sequence consists of 7 nucleotide analoguesand two DNA nucleotides. In some embodiments, the contiguous nucleotidesequence consists of 8 nucleotide analogues and two DNA nucleotides.

The oligomer may consist of the contiguous nucleotide sequence.

In a specially preferred embodiment, all the nucleotide analogues areLNA. In a further preferred embodiment, all nucleotides of the oligomerare LNA. In a further preferred embodiment, all nucleotides of theoligomer are LNA and all internucleoside linkage groups arephosphothioate.

Herein, the term “nitrogenous base” is intended to cover purines andpyrimidines, such as the DNA nucleobases A, C, T and G, the RNAnucleobases A, C, U and G, as well as non-DNA/RNA nucleobases, such as5-methylcytosine (^(Me)C), isocytosine, pseudoisocytosine,5-bromouracil, 5-propynyluracil, 5-propyny-6-fluoroluracil,5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine,2,6-diaminopuine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine and2-chloro-6-aminopurine, in particular ^(Me)C. It will be understood thatthe actual selection of the non-DNA/RNA nucleobase will depend on thecorresponding (or matching) nucleotide present in the microRNA strandwhich the oligonucleotide is intended to target. For example, in casethe corresponding nucleotide is G it will normally be necessary toselect a non-DNA/RNA nucleobase which is capable of establishinghydrogen bonds to G. In this specific case, where the correspondingnucleotide is G, a typical example of a preferred non-DNA/RNA nucleobaseis ^(Me)C.

It should be recognised that the term in ‘one embodiment’ should notnecessarily be limited to refer to one specific embodiment, but mayrefer to a feature which may be present in ‘some embodiments’, or evenas a generic feature of the invention. Likewise, the use of the term‘some embodiments’ may be used to describe a feature of one specificembodiment, or a collection of embodiments, or even as a generic featureof the invention.

The terms “corresponding to” and “corresponds to” refer to thecomparison between the nucleotide sequence of the oligomer or contiguousnucleotide sequence (a first sequence) and the equivalent contiguousnucleotide sequence of a further sequence selected from either i) asub-sequence of the reverse complement of the microRNA nucleic acidtarget (such as a microRNA target selected from SEQ ID 40-SEQ ID 976,and/or ii) the sequence of nucleotides provided herein such as the groupconsisting of SEQ ID NO 977-1913, or SEQ ID NO 1914-2850, or SEQ ID NO2851-3787. Nucleotide analogues are compared directly to theirequivalent or corresponding nucleotides. A first sequence whichcorresponds to a further sequence under i) or ii) typically is identicalto that sequence over the length of the first sequence (such as thecontiguous nucleotide sequence).

When referring to the length of a nucleotide molecule as referred toherein, the length corresponds to the number of monomer units, i.e.nucleotides, irrespective as to whether those monomer units arenucleotides or nucleotide analogues. With respect to nucleotides ornucleobases, the terms monomer and unit are used interchangeably herein.

It should be understood that when the term “about” is used in thecontext of specific values or ranges of values, the disclosure should beread as to include the specific value or range referred to.

As used herein, “hybridisation” means hydrogen bonding, which may beWatson-Crick, Hoogsteen, reversed Hoogsteen hydrogen bonding, etc.,between complementary nucleoside or nucleotide bases. The fournucleobases commonly found in DNA are G, A, T and C of which G pairswith C, and A pairs with T. In RNA T is replaced with uracil (U), whichthen pairs with A. The chemical groups in the nucleobases thatparticipate in standard duplex formation constitute the Watson-Crickface. Hoogsteen showed a couple of years later that the purinenucleobases (G and A) in addition to their Watson-Crick face have aHoogsteen face that can be recognised from the outside of a duplex, andused to bind pyrimidine oligonucleotides via hydrogen bonding, therebyforming a triple helix structure.

In the context of the present invention “complementary” refers to thecapacity for precise pairing between two nucleotides sequences with oneanother. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thecorresponding position of a DNA or RNA molecule, then theoligonucleotide and the DNA or RNA are considered to be complementary toeach other at that position. The DNA or RNA strand are consideredcomplementary to each other when a sufficient number of nucleotides inthe oligonucleotide can form hydrogen bonds with correspondingnucleotides in the target DNA or RNA to enable the formation of a stablecomplex. To be stable in vitro or in vivo the sequence of anoligonucleotide need not be 100% complementary to its target microRNA.The terms “complementary” and “specifically hybridisable” thus implythat the oligonucleotide binds sufficiently strong and specific to thetarget molecule to provide the desired interference with the normalfunction of the target whilst leaving the function of non-target RNAsunaffected. However, in one preferred embodiment the term complementaryshall mean 100% complementary or fully complementary.

In a preferred example the oligonucleotide of the invention is 100%complementary to a miRNA sequence, such as a human microRNA sequence, orone of the microRNA sequences referred to herein.

In a preferred example, the oligonucleotide of the invention comprises acontiguous sequence, which is 100% complementary to the seed region ofthe human microRNA sequence.

Preferably, the term “microRNA” or “miRNA”, in the context of thepresent invention, means an RNA oligonucleotide consisting of between 18to 25 nucleotides in length. In functional terms miRNAs are typicallyregulatory endogenous RNA molecules.

The terms “target microRNA” or “target miRNA” refer to a microRNA with abiological role in human disease, e.g. an upregulated, oncogenic miRNAor a tumor suppressor miRNA in cancer, thereby being a target fortherapeutic intervention of the disease in question.

The terms “target gene” or “target mRNA” refer to regulatory mRNAtargets of microRNAs, in which said “target gene” or “target mRNA” isregulated post-transcriptionally by the microRNA based on near-perfector perfect complementarity between the miRNA and its target siteresulting in target mRNA cleavage; or limited complementarity, oftenconferred to complementarity between the so-called seed sequence(nucleotides 2-7 of the miRNA) and the target site resulting intranslational inhibition of the target mRNA.

In the context of the present invention the oligonucleotide is singlestranded, this refers to the situation where the oligonucleotide is inthe absence of a complementary oligonucleotide—i.e. it is not a doublestranded oligonucleotide complex, such as an siRNA. In one embodiment,the composition according to the invention does not comprise a furtheroligonucleotide which has a region of complementarity with the oligomerof 5 or more, such as 6, 7, 8, 9, or 10 consecutive nucleotides, such aseight or more.

Length

Surprisingly we have found that such short ‘antimiRs’ provide animproved specific inhibition of microRNAs in vivo, whilst retainingremarkable specificity for the microRNA target. A further benefit hasbeen found to be the ability to inhibit several microRNAs simultaneouslydue to the conservation of homologous short sequences between microRNAspecies—such as the seed regions as described herein. According to thepresent invention, it has been found that it is particularlyadvantageous to have short oligonucleotides of 7, 8, 9, 10 nucleotides,such as 7, 8 or 9 nucleotides.

Sequences

The contiguous nucleotide sequence is complementary (such as 100%complementary—i.e. perfectly complementary) to a corresponding region ofa mammalian, human or viral microRNA (miRNA) sequence, preferably ahuman or viral miRNA sequence.

The microRNA sequence may suitably be a mature microRNA. In someembodiments the microRNA may be a microRNA precursor.

The human microRNA sequence may be selected from SEQ ID No 1-558 asdisclosed in WO2008/046911, which are all hereby and specificallyincorporated by reference. As described in WO2008/046911, thesemicroRNAs are associated with cancer.

The viral microRNA sequence may, in some embodiments, be selected fromthe group consisting of Herpes simplex virus 1, Kaposisarcoma-associated herpesvirus, Epstein Barr virus and Humancytomegalovirus.

In one embodiment, the contiguous nucleotide sequence is complementary(such as 100% complementary) to a corresponding region of a miRNAsequence selected from the group of miRNAs listed in table 1. Table 1provides 7mer, 8mer and 9mer oligomers which target human and viralmicroRNAs published in miRBase (Release12.0—http://microma.sanger.ac.uk/sequences/).

In some embodiments, the oligomers according to the invention mayconsist of or comprise a contiguous nucleotide sequence which iscomplementary to a corresponding microRNA sequence selected from thegroup consisting of miR-1, miR-10b, miR-17-3p, miR-18, miR-19a, miR-19b,miR-20, miR-21, miR-34a, miR-93, miR-106a, miR-106b, miR-122, miR-133,miR-134, miR-138, miR-155, miR-192, miR-194, miR-221, miR-222, miR-375.

Therefore, in one embodiment, the miRNA (i.e target miRNA) is selectedfrom the group consisting of miR-1, miR-10b, miR-17-3p, miR-18, miR-19a,miR-19b, miR-20, miR-21, miR-34a, miR-93, miR-106a, miR-106b, miR-122,miR-133, miR-134, miR-138, miR-155, miR-192, miR-194, miR-221, miR-222,and miR-375.

In one embodiment, the miRNA target is a member of the miR 17-92cluster, such as miR 17, miR 106a, miR 106b, miR 18, miR 19a, miR 19b/1,miR 19b/2, miR20/93, miR92/1, miR92/2 and miR25.

In some embodiments the contiguous nucleotide sequence is complementaryto a corresponding region of a microRNA (miRNA) sequence selected fromthe group consisting of miR-21, miR-155, miR-221, mir-222, and mir-122.

In some embodiments said miRNA is selected from the group consisting ofmiR-1, miR-10miR-29, miR-125b, miR-126, miR-133, miR-141, miR-143,miR-200b, miR-206, miR-208, miR-302, miR-372, miR-373, miR-375, andmiR-520c/e.

In some embodiments the contiguous nucleotide sequence is complementaryto a corresponding region of a microRNA (miRNA) sequence present in themiR 17-92 cluster, such as a microRNA selected from the group consistingof miR-17-5p, miR-20a/b, miR-93, miR-106a/b, miR-18a/b, miR-19a/b,miR-25, miR-92a, miR-363.

In one embodiment, the miRNA (i.e target miRNA) is miR-21, such ashsa-miR-21. In one embodiment, the miRNA (i.e target miRNA) is miR-122,such as hsa-miR-122. In one embodiment, the miRNA (i.e target miRNA) ismiR-19b, such as hsa-miR-19b. In one embodiment, the miRNA (i.e targetmiRNA) is miR-155, such as hsa-miR-155. In one embodiment, the miRNA(i.e target miRNA) is miR-375, such as hsa-miR-375. In one embodiment,the miRNA (i.e target miRNA) is miR-375, such as hsa-miR-106b.

Suitably, the contiguous nucleotide sequence may be complementary to acorresponding region of the microRNA, such as a hsa-miR selected fromthe group consisting of 19b, 21, 122, 155 and 375.

The Seed Region and Seedmers

The inventors have found that carefully designed short single strandedoligonucleotides comprising or consisting of nucleotide analogues, suchas high affinity nucleotide analogues such as locked nucleic acid (LNA)units, show significant silencing of microRNAs, resulting in reducedmicroRNA levels. It was found that tight binding of saidoligonucleotides to the so-called seed sequence, typically nucleotides 2to 8 or 2 to 7, counting from the 5′ end, of the target microRNAs wasimportant. Nucleotide 1 of the target microRNAs is a non-pairing baseand is most likely hidden in a binding pocket in the Ago 2 protein.Whilst not wishing to be bound to a specific theory, the presentinventors consider that by selecting the seed region sequences,particularly with oligonucleotides that comprise LNA, preferably LNAunits in the region which is complementary to the seed region, theduplex between miRNA and oligonucleotide is particularly effective intargeting miRNAs, avoiding off target effects, and possibly providing afurther feature which prevents RISC directed miRNA function.

The inventors have found that microRNA silencing is even more enhancedwhen LNA-modified single stranded oligonucleotides do not contain anucleotide at the 3′ end corresponding to this non-paired nucleotide 1.It was further found that at least two LNA units in the 3′ end of theoligonucleotides according to the present invention made saidoligonucleotides highly nuclease resistant.

In one embodiment, the first or second 3′ nucleotide of the oligomercorresponds to the second 5′ nucleotide of the microRNA sequence, andmay be a nucleotide analogue, such as LNA.

In one embodiment, nucleotide units 1 to 6 (inclusive) of the oligomeras measured from the 3′ end the region of the oligomer are complementaryto the microRNA seed region sequence, and may all be nucleotideanalogues, such as LNA.

In one embodiment, nucleotide units 1 to 7 (inclusive) of the oligomeras measured from the 3′ end the region of the oligomer are complementaryto the microRNA seed region sequence, and may all be nucleotideanalogues, such as LNA.

In one embodiment, nucleotide units 2 to 7 (inclusive) of the oligomeras measured from the 3′ end the region of the oligomer are complementaryto the microRNA seed region sequence, and may all be nucleotideanalogues, such as LNA.

In one embodiment, the oligomer comprises at least one nucleotideanalogue unit, such as at least one LNA unit, in a position which iswithin the region complementary to the miRNA seed region. The oligomermay, in one embodiment comprise at between one and 6 or between 1 and 7nucleotide analogue units, such as between 1 and 6 and 1 and 7 LNAunits, in a position which is within the region complementary to themiRNA seed region.

In one embodiment, the contiguous nucleotide sequence consists of orcomprises a sequence which is complementary (such as 100% complementary)to the seed sequence of said microRNA.

In one embodiment, the contiguous nucleotide sequence consists of orcomprises a sequence selected from any one of the seedmer sequenceslisted in table 1.

In one embodiment, the 3′ nucleotide of the seedmer forms the 3′ mostnucleotide of the contiguous nucleotide sequence, wherein the contiguousnucleotide sequence may, optionally, comprise one or two furthernucleotide 5′ to the seedmer sequence.

In one embodiment, the oligomer does not comprise a nucleotide whichcorresponds to the first nucleotide present in the microRNA sequencecounted from the 5′ end.

In one embodiment, the oligonucleotide according to the invention doesnot comprise a nucleotide at the 3′ end that corresponds to the first 5′end nucleotide of the target microRNA.

Nucleotide Analogues

According to the present invention, it has been found that it isparticularly advantageous to have short oligonucleotides of 7, 8, 9, 10nucleotides, such as 7, 8 or 9 nucleotides, wherein at least 50%, suchas 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or such as 100% of thenucleotide units of the oligomer are (preferably high affinity)nucleotide analogues, such as a Locked Nucleic Acid (LNA) nucleotideunit.

In some embodiments, the oligonucleotide of the invention is 7, 8 or 9nucleotides long, and comprises a contiguous nucleotide sequence whichis complementary to a seed region of a human or viral microRNA, andwherein at least 75%, such as at least 80%, such as at least 85%, suchas at least 90%, such as at least 95%, such as 100% of the nucleotidesare are Locked Nucleic Acid (LNA) nucleotide units.

In such oligomers, in some embodiments, the linkage groups are otherthan phosphodiester linkages, such as are phosphorothioate linkages.

In one embodiment, all of the nucleotide units of the contiguousnucleotide sequence are LNA nucleotide units.

In one embodiment, the contiguous nucleotide sequence comprises orconsists of 7, 8, 9 or 10, preferably contiguous, LNA nucleotide units.

In a further preferred embodiment, the oligonucleotide of the inventionis 7, 8 or 9 nucleotides long, and comprises a contiguous nucleotidesequence which is complementary to a seed region of a human or viralmicroRNA, and wherein at least 80% of the nucleotides are LNA, andwherein at least 80%, such as 85%, such as 90%, such as 95%, such as100% of the intemucleotide bonds are phosphorothioate bonds. It will berecognised that the contiguous nucleotide sequence of the oligmer (aseedmer) may extend beyond the seed region.

In some embodiments, the oligonucleotide of the invention is 7nucleotides long, which are all LNA.

In some embodiments, the oligonucleotide of the invention is 8nucleotides long, of which up to 1 nucleotide may be other than LNA. Insome embodiments, the oligonucleotide of the invention is 9 nucleotideslong, of which up to 1 or 2 nucleotides may be other than LNA. In someembodiments, the oligonucleotide of the invention is 10 nucleotideslong, of which 1, 2 or 3 nucleotides may be other than LNA. Thenucleotides ‘other than LNA, may for example, be DNA, or a 2′substituted nucleotide analogues.

High affinity nucleotide analogues are nucleotide analogues which resultin oligonucleotides which has a higher thermal duplex stability with acomplementary RNA nucleotide than the binding affinity of an equivalentDNA nucleotide. This may be determined by measuring the T_(m).

In some embodiments, the nucleotide analogue units present in thecontiguous nucleotide sequence are selected, optionally independently,from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit,2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INAunit, and a 2′MOE RNA unit.

In some embodiments, the nucleotide analogue units present in thecontiguous nucleotide sequence are selected, optionally independently,from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit,2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, and a 2′MOE RNA unit.

The term 2′fluoro-DNA refers to a DNA analogue with a substitution tofluorine at the 2′ position (2′F). 2′fluoro-DNA is a preferred form of2′fluoro-nucleotide.

In some embodiments, the oligomer comprises at least 4 nucleotideanalogue units, such as at least 5 nucleotide analogue units, such as atleast 6 nucleotide analogue units, such as at least 7 nucleotideanalogue units, such as at least 8 nucleotide analogue units, such as atleast 9 nucleotide analogue units, such as 10, nucleotide analogueunits.

In one embodiment, the oligomer comprises at least 3 LNA units, such asat least 4 LNA units, such as at least 5 LNA units, such as at least 6LNA units, such as at least 7 LNA units, such as at least 8 LNA units,such as at least 9 LNA units, such as 10 LNA.

In one embodiment wherein at least one of the nucleotide analogues, suchas LNA units, is either cytosine or guanine, such as between 1-10 of theof the nucleotide analogues, such as LNA units, is either cytosine orguanine, such as 2, 3, 4, 5, 6, 7, 8, or 9 of the of the nucleotideanalogues, such as LNA units, is either cytosine or guanine.

In one embodiment at least two of the nucleotide analogues such as LNAunits are either cytosine or guanine. In one embodiment at least threeof the nucleotide analogues such as LNA units are either cytosine orguanine. In one embodiment at least four of the nucleotide analoguessuch as LNA units are either cytosine or guanine. In one embodiment atleast five of the nucleotide analogues such as LNA units are eithercytosine or guanine. In one embodiment at least six of the nucleotideanalogues such as LNA units are either cytosine or guanine. In oneembodiment at least seven of the nucleotide analogues such as LNA unitsare either cytosine or guanine. In one embodiment at least eight of thenucleotide analogues such as LNA units are either cytosine or guanine.

In a preferred embodiment the nucleotide analogues have a higher thermalduplex stability for a complementary RNA nucleotide than the bindingaffinity of an equivalent DNA nucleotide to said complementary RNAnucleotide.

In one embodiment, the nucleotide analogues confer enhanced serumstability to the single stranded oligonucleotide.

Whilst the specific SEQ IDs in the sequence listing and table 1 refer tooligomers of LNA monomers with phosphorothioate (PS) backbone, it willbe recognised that the invention also encompasses the use of othernucleotide analogues and/or linkages, either as an alternative to, or incombination with LNA. As such, the sequence of nucleotides (bases) shownin the sequence listings may be of LNA such as LNA/PS, LNA or may beoligomers containing alternative backbone chemistry, such assugar/linkage chemistry, whilst retaining the same base sequence (A, T,C or G).

Whilst it is envisaged that other nucleotide analogues, such as 2′-MOERNA or 2′-fluoro nucleotides may be useful in the oligomers according tothe invention, it is preferred that the oligomers have a highproportion, such as at least 50%, LNA. nucleotides.

The nucleotide analogue may be a DNA analogue such as a DNA analoguewhere the 2′-H group is substituted with a substitution other than —OH(RNA) e.g. by substitution with —O—CH₃, —O—CH₂—CH₂—O—CH₃,—O—CH₂—CH₂—CH₂—NH₂, —O—CH₂—CH₂—CH₂—OH or —F. The nucleotide analogue maybe a RNA analogues such as a RNA analogue which have been modified inits 2′-OH group, e.g. by substitution with a group other than —H (DNA),for example —O—CH₃, —O— CH₂—CH₂—O—CH₃, —O—CH₂—CH₂—CH₂—NH₂,—O—CH₂—CH₂—CH₂—OH or —F. In one embodiment the nucleotide analogue is“ENA”.

LNA

When used in the present context, the terms “LNA unit”, “LNA monomer”,“LNA residue”, “locked nucleic acid unit”, “locked nucleic acid monomer”or “locked nucleic acid residue”, refer to a bicyclic nucleosideanalogue. LNA units are described in inter alia WO 99/14226, WO00/56746, WO 00/56748, WO 01/25248, WO 02/28875, WO 03/006475 and WO03/095467. The LNA unit may also be defined with respect to its chemicalformula. Thus, an “LNA unit”, as used herein, has the chemical structureshown in Scheme 1 below:

wherein

-   -   X is selected from the group consisting of O, S and NR^(H),        where R^(H) is H or C₁₋₄-alkyl; Y is (—CH₂)_(r), where r is an        integer of 1-4; and B is a nitrogenous base.

In a preferred embodiment of the invention, r is 1 or 2, in particular1, i.e. a preferred LNA unit has the chemical structure shown in Scheme2 below:

wherein X and B are as defined above.

In an interesting embodiment, the LNA units incorporated in theoligonucleotides of the invention are independently selected from thegroup consisting of thio-LNA units, amino-LNA units and oxy-LNA units.

Thus, the thio-LNA unit may have the chemical structure shown in Scheme3 below:

wherein B is as defined above.

Preferably, the thio-LNA unit is in its beta-D-form, i.e. having thestructure shown in 3A above. likewise, the amino-LNA unit may have thechemical structure shown in Scheme 4 below:

wherein B and R^(H) are as defined above.

Preferably, the amino-LNA unit is in its beta-D-form, i.e. having thestructure shown in 4A above.

The oxy-LNA unit may have the chemical structure shown in Scheme 5below:

wherein B is as defined above.

Preferably, the oxy-LNA unit is in its beta-D-form, i.e. having thestructure shown in 5A above. As indicated above, B is a nitrogenous basewhich may be of natural or non-natural origin. Specific examples ofnitrogenous bases include adenine (A), cytosine (C), 5-methylcytosine(^(Me)C), isocytosine, pseudoisocytosine, guanine (G), thymine (T),uracil (U), 5-bromouracil, 5-propynyluracil, 5-propyny-6,5-methytthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine,2,6-diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanineand 2-chloro-6-aminopurine.

The term “thio-LNA unit” refers to an LNA unit in which X in Scheme 1 isS. A thio-LNA unit can be in both the beta-D form and in the alpha-Lform. Generally, the beta-D form of the thio-LNA unit is preferred. Thebeta-D-form and alpha-L-form of a thio-LNA unit are shown in Scheme 3 ascompounds 3A and 3B, respectively.

The term “amino-LNA unit” refers to an LNA unit in which X in Scheme 1is NH or NR^(H), where R^(H) is hydrogen or C₁₋₄-alkyl. An amino-LNAunit can be in both the beta-D form and in the alpha-L form. Generally,the beta-D form of the amino-LNA unit is preferred. The beta-D-form andalpha-L-form of an amino-LNA unit are shown in Scheme 4 as compounds 4Aand 4B, respectively.

The term “oxy-LNA unit” refers to an LNA unit in which X in Scheme 1 isO. An Oxy-LNA unit can be in both the beta-D form and in the alpha-Lform. Generally, the beta-D form of the oxy-LNA unit is preferred. Thebeta-D form and the alpha-L form of an oxy-LNA unit are shown in Scheme5 as compounds 5A and 5B, respectively.

In the present context, the term “C₁₋₆-alkyl” is intended to mean alinear or branched saturated hydrocarbon chain wherein the longestchains has from one to six carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl and hexyl. A branched hydrocarbon chain is intendedto mean a C₁₋₆-alkyl substituted at any carbon with a hydrocarbon chain.

In the present context, the term “C₁₋₄-alkyl” is intended to mean alinear or branched saturated hydrocarbon chain wherein the longestchains has from one to four carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Abranched hydrocarbon chain is intended to mean a C₁₋₄-alkyl substitutedat any carbon with a hydrocarbon chain.

When used herein the term “C₁₋₆-alkoxy” is intended to meanC₁₋₆-alkyl-oxy, such as methoxy, ethoxy, n-propoxy, isopropoxy,n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy,neopentoxy and hexoxy.

In the present context, the term “C₂₋₆-alkenyl” is intended to mean alinear or branched hydrocarbon group having from two to six carbon atomsand containing one or more double bonds. Illustrative examples ofC₂₋₆-alkenyl groups include allyl, homo-allyl, vinyl, crotyl, butenyl,butadienyl, pentenyl, pentadienyl, hexenyl and hexadienyl. The positionof the unsaturation (the double bond) may be at any position along thecarbon chain.

In the present context the term “C₂₋₆-alkynyl” is intended to meanlinear or branched hydrocarbon groups containing from two to six carbonatoms and containing one or more triple bonds. Illustrative examples ofC₂₋₆-alkynyl groups include acetylene, propynyl, butynyl, pentynyl andhexynyl. The position of unsaturation (the triple bond) may be at anyposition along the carbon chain. More than one bond may be unsaturatedsuch that the “C₂₋₆-alkynyl” is a di-yne or enedi-yne as is known to theperson skilled in the art.

When referring to substituting a DNA unit by its corresponding LNA unitin the context of the present invention, the term “corresponding LNAunit” is intended to mean that the DNA unit has been replaced by an LNAunit containing the same nitrogenous base as the DNA unit that it hasreplaced, e.g. the corresponding LNA unit of a DNA unit containing thenitrogenous base A also contains the nitrogenous base A. The exceptionis that when a DNA unit contains the base C, the corresponding LNA unitmay contain the base C or the base ^(Me)C, preferably ^(Me)C.

Herein, the term “non-LNA unit” refers to a nucleoside different from anLNA-unit, i.e. the term “non-LNA unit” includes a DNA unit as well as anRNA unit. A preferred non-LNA unit is a DNA unit.

The terms “unit”, “residue” and “monomer” are used interchangeablyherein.

The term “at least one” encompasses an integer larger than or equal to1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 and so forth.

The terms “a” and “an” as used about a nucleotide, an agent, an LNAunit, etc., is intended to mean one or more. In particular, theexpression “a component (such as a nucleotide, an agent, an LNA unit, orthe like) selected from the group consisting of . . . ” is intended tomean that one or more of the cited components may be selected. Thus,expressions like “a component selected from the group consisting of A, Band C” is intended to include all combinations of A, B and C, i.e. A, B,C, A+B, A+C, B+C and A+B+C.

Internucleoside Linkages

The term “internucleoside linkage group” is intended to mean a groupcapable of covalently coupling together two nucleotides, such as betweenDNA units, between DNA units and nucleotide analogues, between twonon-LNA units, between a non-LNA unit and an LNA unit, and between twoLNA units, etc. Examples include phosphate, phosphodiester groups andphosphorothioate groups.

In some embodiments, at least one of, such as all of the internucleosidelinkage in the oligomer is phosphodiester. However for in vivo use,phosphorothioate linkages may be preferred.

Typical internucleoside linkage groups in oligonucleotides are phosphategroups, but these may be replaced by internucleoside linkage groupsdiffering from phosphate. In a further interesting embodiment of theinvention, the oligonucleotide of the invention is modified in itsinternucleoside linkage group structure, i.e. the modifiedoligonucleotide comprises an internucleoside linkage group which differsfrom phosphate. Accordingly, in a preferred embodiment, theoligonucleotide according to the present invention comprises at leastone internucleoside linkage group which differs from phosphate.

Specific examples of internucleoside linkage groups which differ fromphosphate

(—O—P(O)₂—O—) include —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—,—S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—,—O—PO(R^(H))—O—, O—PO(OCH₃)—O—, —O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—,—O—PO(BH₃)—O—, —O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—,—NR^(H)—CO—O—, —NR^(H)—CO—NR^(H)—, —O—CO—O—, —O—CO—NR^(H)—,—NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H), —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—,—CH₂—NR^(H)—CO—, —O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—,—CH₂—SO₂—CH₂, —CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR—CO—, —CH₂—NCH₃—O—CH₂—,where R^(H) is hydrogen or C₁₋₄-alkyl.

When the internucleoside linkage group is modified, the internucleosidelinkage group is preferably a phosphorothioate group (—O—P(O,S)—O—). Ina preferred embodiment, all internucleoside linkage groups of theoligonucleotides according to the present invention arephosphorothioate.

The internucleoside linkage may be selected form the group consistingof: —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—,—S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—,O—PO(OCH₃)—O—, —O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,—NR^(H)—CO—NR^(H)—, and/or the internucleoside linkage may be selectedform the group consisting of: —O—CO—CO—O—CO—NR^(H)—, —NR^(H)—CO—CH₂—,—O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—, —CH₂—NR^(H)—CO—,—O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—SO₂—CH₂—,—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—CO—, —CH₂—NCH₃—O—CH₂—, where R^(H) isselected from hydrogen and C₁₋₄-alkyl. Suitably, in some embodiments,sulphur (S) containing internucleoside linkages as provided above may bepreferred. The internucleoside linkages may be independently selected,or all be the same, such as phosphorothioate linkages.

In one embodiment, at least 75%, such as 80% or 85% or 90% or 95% or allof the internucleoside linkages present between the nucleotide units ofthe contiguous nucleotide sequence are phosphorothioate internucleosidelinkages.

Micromir Oligonucleotides Targeting More than One microRNA

In one embodiment, the contiguous nucleotide sequence is complementaryto the corresponding sequence of at least two miRNA sequences such as 2,3, 4, 5, 6, 7, 8, 9, or 10 miRNA sequence. The use of a single universalbase may allow a single oligomer of the invention to target twoindependent microRNAs which either one or both have a single mismatch inthe region which corresponds to oligomer at the position where theuniversal nucleotide is positioned.

In one embodiment, the contiguous nucleotide sequence consists of orcomprises a sequence which is complementary to the sequence of at leasttwo miRNA seed region sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10miRNA seed region sequences.

In one embodiment, the contiguous nucleotide sequence is complementaryto the corresponding region of both miR-221 and miR-222.

In one embodiment, the contiguous nucleotide sequence is complementaryto the corresponding region of more than one member of the miR-17-92cluster—such as two or more or all of miR-17-5p, miR-20a/b, miR-93,miR-106a/b; or two or more or all of miR-25, miR-92a and miR-363.

In one embodiment, the contiguous nucleotide sequence consists of orcomprises a sequence that is complementary to 5′GCTACAT3′.

Oligomer Design

In one embodiment, the first nucleotide of the oligomer according to theinvention, counting from the 3′ end, is a nucleotide analogue, such asan LNA unit. In one embodiment, which may be the same or different, thelast nucleotide of the oligomer according to the invention, countingfrom the 3′ end, is a nucleotide analogue, such as an LNA unit.

In one embodiment, the second nucleotide of the oligomer according tothe invention, counting from the 3′ end, is a nucleotide analogue, suchas an LNA unit.

In one embodiment, the ninth and/or the tenth nucleotide of the oligomeraccording to the invention, counting from the 3′ end, is a nucleotideanalogue, such as an LNA unit.

In one embodiment, the ninth nucleotide of the oligomer according to theinvention, counting from the 3′ end is a nucleotide analogue, such as anLNA unit.

In one embodiment, the tenth nucleotide of the oligomer according to theinvention, counting from the 3′ end is a nucleotide analogue, such as anLNA unit.

In one embodiment, both the ninth and the tenth nucleotide of theoligomer according to the invention, calculated from the 3′ end is anucleotide analogue, such as an LNA unit.

In one embodiment, the oligomer according to the invention does notcomprise a region of more than 3 consecutive DNA nucleotide units. Inone embodiment, the oligomer according to the invention does notcomprise a region of more than 2 consecutive DNA nucleotide units.

In one embodiment, the oligomer comprises at least a region consistingof at least two consecutive nucleotide analogue units, such as at leasttwo consecutive LNA units. In one embodiment, the oligomer comprises atleast a region consisting of at least three consecutive nucleotideanalogue units, such as at least three consecutive LNA units.

Other Patterns of Nucleotide Analogues Such as LNA in the Oligomer

Whilst it is envisaged that oligomers containing at least 6 LNA, such asat least 7 nucleotide units may be preferable, the discovery that suchshort oligomers are highly effective at targeting microRNAs in vivo canbe used to prepare shorter oligomers of the invention which compriseother nucleotide analogues, such as high affinity nucleotide analogues.Indeed, the combination of LNA with other high affinity nucleotideanalogues are considered as part of the present invention.

Modification of nucleotides in positions 1 to 2, counting from the 3′end. The nucleotide at positions 1 and/or 2 may be a nucleotideanalogue, such as a high affinity nucleotide analogue, such as LNA, or anucleotide analogue selected from the group consisting of 2′-O-alkyl-RNAunit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNAunit, LNA unit, PNA unit, HNA unit, INA unit. The two 3′ nucleotide maytherefore be

Xx, xX, XX or xx, wherein: In one embodiment X is LNA and x is DNA oranother nucleotide analogue, such as as a 2′ substituted nucleotideanalogue selected from the group consisting of 2′-O_alkyl-RNA unit,2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOERNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or2′-fluoro-DNA. Alternatively X is a nucleotide analogue, and x is DNA.

The above modification at the 2 3′ terminal nucleotides may be combinedwith modification of nucleotides in positions 3-8 counting from the 3′end, as described below. In this respect nucleotides designated as X andx may be the same throughout the oligomer. It will be noted that whenthe oligomer is only 7 nucleotides in length the 8^(th) nucleotidecounting from the 3′ end should be discarded. In the followingembodiments which refer to the modification of nucleotides in positions3 to 8, counting from the 3′ end, the LNA units, in one embodiment, maybe replaced with other nucleotide anlogues, such as those referred toherein. “X” may, therefore be selected from the group consisting of2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNAunit, 2′-MOE-RNA unit, LNA unit, PNA unit, HNA unit, INA unit. “x” ispreferably DNA or RNA, most preferably DNA. However, it is preferredthat X is LNA.

In one embodiment of the invention, the oligonucleotides of theinvention are modified in positions 3 to 8, counting from the 3′ end.The design of this sequence may be defined by the number of non-LNAunits present or by the number of LNA units present. In a preferredembodiment of the former, at least one, such as one, of the nucleotidesin positions three to eight, counting from the 3′ end, is a non-LNAunit. In another embodiment, at least two, such as two, of thenucleotides in positions three to eight, counting from the 3′ end, arenon-LNA units. In yet another embodiment, at least three, such as three,of the nucleotides in positions three to eight, counting from the 3′end, are non-LNA units. In still another embodiment, at least four, suchas four, of the nucleotides in positions three to eight, counting fromthe 3′ end, are non-LNA units. In a further embodiment, at least five,such as five, of the nucleotides in positions three to eight, countingfrom the 3′ end, are non-LNA units. In yet a further embodiment, all sixnucleotides in positions three to eight, counting from the 3′ end, arenon-LNA units.

Alternatively defined, in an embodiment, the oligonucleotide accordingto the present invention comprises at least three LNA units in positionsthree to eight, counting from the 3′ end.

In an embodiment thereof, the oligonucleotide according to the presentinvention comprises three LNA units in positions three to eight,counting from the 3′ end. The substitution pattern for the nucleotidesin positions three to eight, counting from the 3′ end, may be selectedfrom the group consisting of XXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxXxx,XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx,xXxxXX, xxXxXX, xXxXxX and XxXxXx, wherein “X” denotes an LNA unit and“x” denotes a non-LNA unit. In a preferred embodiment, the substitutionpattern for the nucleotides in positions three to eight, counting fromthe 3′ end, is selected from the group consisting of XXxXxx, XXxxXx,XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX,xxXxXX, xXxXxX and XxXxXx, wherein “X” denotes an LNA unit and “x”denotes a non-LNA unit. In a more preferred embodiment, the substitutionpattern for the nucleotides in positions three to eight, counting fromthe 3′ end, is selected from the group consisting of xXXxXx, xXXxxX,xxXXxX, xXxXXx, xXxxXX, xxXxXX and xXxXxX, wherein “X” denotes an LNAunit and “x” denotes a non-LNA unit. In an embodiment, the substitutionpattern for the nucleotides in positions three to eight, counting fromthe 3′ end, is xXxXxX or XxXxXx, wherein “X” denotes an LNA unit and “x”denotes a non-LNA unit. In an embodiment, the substitution pattern forthe nucleotides in positions three to eight, counting from the 3′ end,is xXxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNAunit.

In a further embodiment, the oligonucleotide according to the presentinvention comprises at least four LNA units in positions three to eight,counting from the 3′ end. In an embodiment thereof, the oligonucleotideaccording to the present invention comprises four LNA units in positionsthree to eight, counting from the 3′ end. The substitution pattern forthe nucleotides in positions three to eight, counting from the 3′ end,may be selected from the group consisting of xxXXXX, xXxXXX, xXXxXX,xXXXxX, xXXXXx, XxxXXX, XxXxXX, XxXXxX, XxXXXx, XXxxXX, XXxXxX, XXxXXx,XXXxxX, XXXxXx and XXXXxx, wherein “X” denotes an LNA unit and “x”denotes a non-LNA unit.

In yet a further embodiment, the oligonucleotide according to thepresent invention comprises at least five LNA units in positions threeto eight, counting from the 3′ end. In an embodiment thereof, theoligonucleotide according to the present invention comprises five LNAunits in positions three to eight, counting from the 3′ end. Thesubstitution pattern for the nucleotides in positions three to eight,counting from the 3′ end, may be selected from the group consisting ofxXXXXX, XxXXXX, XXxXXX, XXXxXX, XXXXxX and XXXXXx, wherein “X” denotesan LNA unit and “x” denotes a non-LNA unit.

Preferably, the oligonucleotide according to the present inventioncomprises one or two LNA units in positions three to eight, countingfrom the 3′ end. This is considered advantageous for the stability ofthe A-helix formed by the oligo:microRNA duplex, a duplex resembling anRNA:RNA duplex in structure.

In yet a further embodiment, the oligonucleotide according to thepresent invention comprises at least six LNA units in positions three toeight, counting from the 3′ end. In an embodiment thereof, theoligonucleotide according to the present invention comprises at fromthree to six LNA units in positions three to eight, counting from the 3′end, and in addition from none to three other high affinity nucleotideanalogues in the same region, such that the total amount of highaffinity nucleotide analogues (including the LNA units) amount to six inthe region from positions three to eight, counting from the 3′ end.

In some embodiments, such as when X is LNA, said non-LNA unit (x) isanother nucleotide analogue unit, such as a 2′ substituted nucleotideanalogue selected from the group consisting of 2′-O_alkyl-RNA unit,2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOERNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or2′-fluoro-DNA.

For oligomers which have 9 or 10 nucleotides, the nucleotide atpositions 9 and/or 10 may be a nucleotide analogue, such as a highaffinity nucleotide analogue, such as LNA, or a nucleotide analogueselected from the group consisting of 2′-O-alkyl-RNA unit, 2′-OMe-RNAunit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNA unit, LNA unit,PNA unit, HNA unit, INA unit. The two 5′ nucleotides may therefore be

Xx, xX, XX or xx, wherein: In one embodiment X is LNA and x is DNA oranother nucleotide analogue, such as as a 2′ substituted nucleotideanalogue selected from the group consisting of 2′-O_alkyl-RNA unit,2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOERNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or2′-fluoro-DNA. Alternatively X is a nucleotide analogue, and x is DNA.

The above modification at the 2 5′ terminal nucleotides may be combinedwith modification of nucleotides in positions 3-8 counting from the 3′end, and/or the 2 3′ nucleotitides as described above. In this respectnucleotides designated as X and x may be the same throughout theoligomer.

In a preferred embodiment of the invention, the oligonucleotideaccording to the present invention contains an LNA unit at the 5′ end.In another preferred embodiment, the oligonucleotide according to thepresent invention contains an LNA unit at the first two positions,counting from the 5′ end.

In one embodiment, the invention further provides for an oligomer asdescribed in the context of the pharmaceutical composition of theinvention, or for use in vivo in an organism, such as a medicament,wherein said oligomer (or contiguous nucleotide sequence) compriseseither

-   -   i) at least one phosphorothioate linkage and/or    -   ii) at least one 3′ terminal LNA unit, and/or    -   iii) at least one 5′ terminal LNA unit.

The oligomer may therefore contain at least one phosphorothioatelinkage, such as all linkages being phosphorthioates, and at least one3′ terminal LNA unit, and at least one 5′ terminal LNA unit.

It is preferable for most therapeutic uses that the oligonucleotide isfully phosphorothiolated—an exception being for therapeuticoligonucleotides for use in the CNS, such as in the brain or spine wherephosphorothioation can be toxic, and due to the absence of nucleases,phosphodiester bonds may be used, even between consecutive DNA units.

As referred to herein, other in one aspect of the oligonucleotideaccording to the invention is that the second 3′ nucleotide, and/or the9^(t) and 10 (from the 3′ end), if present, may also be LNA.

In one embodiment, the oligomer comprises at least five nucleotideanalogue units, such as at least five LNA units, in positions which arecomplementary to the miRNA seed region.

In one embodiment, the nucleotide sequence of the oligomer which iscomplementary to the sequence of the microRNA seed region, is selectedfrom the group consisting of (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX,(X)XXXXxX and (X)XXXXXx, wherein “X” denotes a nucleotide analogue, suchas an LNA unit, (X) denotes an optional nucleotide analogue, such as anLNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the oligomer comprises six or seven nucleotideanalogue units, such as six or seven LNA units, in positions which arecomplementary to the miRNA seed region.

In one embodiment, the nucleotide sequence of the oligomer which iscomplementary to the sequence of the microRNA seed region, is selectedfrom the group consisting of XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX,XXXXXxX and XXXXXXx, wherein “X” denotes a nucleotide analogue, such asan LNA unit, such as an LNA unit, and “x” denotes a DNA or RNAnucleotide unit.

In one embodiment, the two nucleotide motif at position 7 to 8, countingfrom the 3′ end of the oligomer is selected from the group consisting ofxx, XX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as anLNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotideunit.

In one embodiment, the two nucleotide motif at position 7 to 8, countingfrom the 3′ end of the oligomer is selected from the group consisting ofXX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as an LNAunit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the oligomer comprises at 12 nucleotides and whereinthe two nucleotide motif at position 11 to 12, counting from the 3′ endof the oligomer is selected from the group consisting of xx, XX, xX andXx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, suchas an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the oligomer comprises 12 nucleotides and wherein thetwo nucleotide motif at position 11 to 12, counting from the 3′ end ofthe oligomer is selected from the group consisting of XX, xX and Xx,wherein “X” denotes a nucleotide analogue, such as an LNA unit, such asan LNA unit, and “x” denotes a DNA or RNA nucleotide unit, such as a DNAunit.

In one embodiment, the oligomer comprises a nucleotide analogue unit,such as an LNA unit, at the 5′ end.

In one embodiment, the nucleotide analogue units, such as X, areindependently selected form the group consisting of: 2′-O-alkyl-RNAunit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNAunit, LNA unit, PNA unit, HNA unit, INA unit.

In one embodiment, all the nucleotides of the oligomer of the inventionare nucleotide analogue units.

In one embodiment, the nucleotide analogue units, such as X, areindependently selected form the group consisting of: 2′-OMe-RNA units,2′-fluoro-DNA units, and LNA units,

In one embodiment, the oligomer comprises said at least one LNA analogueunit and at least one further nucleotide analogue unit other than LNA.

In one embodiment, the non-LNA nucleotide analogue unit or units areindependently selected from 2′-OMe RNA units and 2′-fluoro DNA units.

In one embodiment, the oligomer consists of at least one sequence XYX orYXY, wherein X is LNA and Y is either a 2′-OMe RNA unit and 2′-fluoroDNA unit.

In one embodiment, the sequence of nucleotides of the oligomer consistsof alternative X and Y units.

In one embodiment, the oligomer comprises alternating LNA and DNA units(Xx) or (xX). In one embodiment, the oligomer comprises a motif ofalternating LNA followed by 2 DNA units (Xxx), xXx or xxX.

In one embodiment, at least one of the DNA or non-LNA nucleotideanalogue units are replaced with a LNA nucleotide in a position selectedfrom the positions identified as LNA nucleotide units in any one of theembodiments referred to above. In one embodiment, “X” donates an LNAunit.

Further Designs for Oligomers of the Invention

Table 1 below provides non-limiting examples of short microRNA sequencesthat could advantageously be targeted with an oligonucleotide of thepresent invention.

The oligonucleotides according to the invention, such as those disclosedin table 1 may, in one embodiment, have a sequence of 7, 8, 9 or 10 LNAnucleotides 5′-3′ LLLLLLL(L)(L)(L)(L), or have a sequence of nucleotidesselected form the group consisting of, the first 7, 8, 9 or 10nucleotides of the following motifs:

LdLddL(L)(d)(d)(L)(d)(L)(d)(L)(L), LdLdLL(L)(d)(d)(L)(L)(L)(d)(L)(L),LMLMML(L)(M)(M)(L)(M)(L)(M)(L)(L), LMLMLL(L)(M)(M)(L)(L)(L)(M)(L)(L),LFLFFL(L)(F)(F)(L)(F)(L)(F)(L)(L), LFLFLL(L)(F)(F)(L)(L)(L)(F)(L)(L),and every third designs such as; LddLdd(L)(d)(d)(L)(d)(d)(L)(d)(d)(L)(d)′dLddLd(d)(L)(d)(d)(L)(d)(d)(L)(d)(d)(L),ddLddL(d)(d)(L)(d)(d)(L)(d)(d)(L)(d)(d),LMMLMM(L)(M)(M)(L)(M)(M)(L)(M)(M)(L)(M),MLMMLM(M)(L)(M)(M)(L)(M)(M)(L)(M)(M)(L),MMLMML(M)(M)(L)(M)(M)(L)(M)(M)(L)(M)(M),LFFLFF(L)(F)(F)(L)(F)(F)(L)(F)(F)(L)(F),FLFFLF(F)(L)(F)(F)(L)(F)(F)(L)(F)(F)(L),FFLFFL(F)(F)(L)(F)(F)(L)(F)(F)(L)(F)(F), anddLdLdL(d)(L)(d)(L)(d)(L)(d)(L)(d)(L)(d) and an every second design, suchas; LdLdLd(L)(d)(L)(d)(L)(d)(L)(d)(L)(d)(L),MLMLML(M)(L)(M)(L)(M)(L)(M)(L)(M)(L)(M),LMLMLM(L)(M)(L)(M)(L)(M)(L)(M)(L)(M)(L),FLFLFL(F)(L)(F)(L)(F)(L)(F)(L)(F)(L)(F), andLFLFLF(L)(F)(L)(F)(L)(F)(L)(F)(L)(F)(L); wherein L=LNA unit, d=DNAunits, M=2′MOE RNA, F=2′Fluoro and residues in brackets are optional.

Pharmaceutical Composition and Medical Application

The invention provides for a pharmaceutical composition comprising theoligomer according to the invention, and a pharmaceutically acceptablediluent, carrier, salt or adjuvant.

The invention further provides for the use of an oligonucleotideaccording to the invention, such as those which may form part of thepharmaceutical composition, for the manufacture of a medicament for thetreatment of a disease or medical disorder associated with the presenceor over-expression (upregulation) of the microRNA.

The invention further provides for a method for the treatment of adisease or medical disorder associated with the presence orover-expression of the microRNA, comprising the step of administering acomposition (such as the pharmaceutical composition) according to theinvention to a person in need of treatment.

The invention further provides for a method for reducing the effectiveamount of a miRNA in a cell or an organism, comprising administering acomposition (such as the pharmaceutical composition) according to theinvention or a oligomer according to the invention to the cell or theorganism. Reducing the effective amount in this context refers to thereduction of functional miRNA present in the cell or organism. It isrecognised that the preferred oligonucleotides according to theinvention may not always significantly reduce the actual amount of miRNAin the cell or organism as they typically form very stable duplexes withtheir miRNA targets. The reduction of the effective amount of the miRNAin a cell may, in one embodiment, be measured by detecting the level ofde-repression of the miRNA's target in the cell.

The invention further provides for a method for de-repression of atarget mRNA of a miRNA in a cell or an organism, comprisingadministering a composition (such as the pharmaceutical composition) ora oligomer according to the invention to the cell or the organism.

The invention further provides for the use of a oligomer of between 7-10such as 7, 8, 9, or 10 nucleotides in length, for the manufacture of amedicament for the treatment of a disease or medical disorder associatedwith the presence or over-expression of the microRNA.

In one embodiment the medical condition (or disease) is hepatitis C(HCV), and the miRNA is miR-122.

In one embodiment, the pharmaceutical composition according to theinvention is for use in the treatment of a medical disorder or diseaseselected from the group consisting of: hepatitis C virus infection andhypercholesterolemia and related disorders, and cancers.

In one embodiment the medical disorder or disease is a CNS disease, suchas a CNS disease where one or more microRNAs are known to be indicated.

In the context of hypercholesterolemia related disorders refers todiseases such as atherosclerosis or hyperlipidemia. Further examples ofrelated diseases also include different types of HDL/LDL cholesterolimbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL),acquired hyperlipidemia, statin-resistant hypercholesterolemia; coronaryartery disease (CAD) coronary heart disease (CHD), atherosclerosis.

In one embodiment, the pharmaceutical composition according to theinvention further comprises a second independent active ingredient thatis an inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor,or an MTP inhibitor (such as those disclosed in U.S. 60/977,497, herebyincorporated by reference).

The invention further provides for a method for the treatment of adisease or medical disorder associated with the presence orover-expression of the microRNA, comprising the step of administering acomposition (such as the pharmaceutical composition) comprising aoligomer of between between 7-10 such as 7, 8, 9, or 10 nucleotides inlength, to a person in need of treatment.

The invention further provides for a method for reducing the effectiveamount of a miRNA target (i.e. ‘available’ miRNA) in a cell or anorganism, comprising administering a composition (such as thepharmaceutical composition) comprising a oligomer of between 6 7-10 suchas 7, 8, 9, or 10 nucleotides in length, to the cell or the organism.

It should be recognised that “reducing the effective amount” of one ormore microRNAs in a cell or organism, refers to the inhibition of themicroRNA function in the call or organism. The cell is preferablyamammalain cell or a human cell which expresses the microRNA ormicroRNAs.

The invention further provides for a method for de-repression of atarget mRNA of a miRNA in a cell or an organism, comprising a oligomerof 7-10 such as 7, 8, 9, or 10 nucleotides in length, or (or acomposition comprising said oligonucleotide) to the cell or theorganism.

As mentioned above, microRNAs are related to a number of diseases.Hence, a fourth aspect of the invention relates to the use of anoligonucleotide as defined herein for the manufacture of a medicamentfor the treatment of a disease associated with the expression ofmicroRNAs selected from the group consisting of spinal muscular atrophy,Tourette's syndrome, hepatitis C, fragile X mental retardation, DiGeorgesyndrome and cancer, such as in non limiting example, chroniclymphocytic leukemia, breast cancer, lung cancer and colon cancer, inparticular cancer.

Methods of Synthesis

The invention further provides for a method for the synthesis of anoligomer targeted against a human microRNA, such as an oligomerdescribed herein, said method comprising the steps of:

-   -   a. Optionally selecting a first nucleotide, counting from the 3′        end, which is a nucleotide analogue, such as an LNA nucleotide.    -   b. Optionally selecting a second nucleotide, counting from the        3′ end, which is a nucleotide analogue, such as an LNA        nucleotide.    -   c. Selecting a region of the oligomer which corresponds to the        miRNA seed region, wherein said region is as defined herein.    -   d. Selecting a seventh and optionally an eight nucleotideas        defined herein.    -   e. Optionally selecting one or two further 5′ terminal of the        oligomer is as defined herein;

wherein the synthesis is performed by sequential synthesis of theregions defined in steps a-e, wherein said synthesis may be performed ineither the 3′-5′ (a to f) or 5′-3′ (e to a) direction, and wherein saidoligomer is complementary to a sequence of the miRNA target.

The invention further provides for a method for the preparation of anoligomer (such as an oligomer according to the invention), said methodcomprising the steps of a) comparing the sequences of two or more miRNAsequences to identify two or more miRNA sequences which comprise acommon contiguous nucleotide sequence of at least 7 nucleotides inlength, such as 7, 8, 9 or 10 nucleotides in length (i.e. a sequencefound in both non-identical miRNAs), b) preparing an oligomer sequencewhich consists or comprises of a contiguous nucleotide sequence with iscomplementary to said common contiguous nucleotide sequence, whereinsaid oligomer is, as according to the oligomer of the invention. In apreferred example, the common contiguous nucleotide sequence consists orcomprises of the seed region of each of said two or more miRNA sequences(which comprise a common contiguous nucleotide sequence of at least 6nucleotides in length). In one embodiment, the seed regions of the twoor more miRNAs are identical. Suitably the oligomer consists orcomprises a seedmer sequence of 7 or 8 nucleotides in length whichcomprises of a sequence which is complementary to said two or moremiRNAs. This method may be used in conjunction with step c of the abovemethod.

The method for the synthesis of the oligomer according to the inventionmay be performed using standard solid phase oligonucleotide systhesis.

In one embodiment, the method for the synthesis of a oligomer targetedagainst a human microRNA, is performed in the 3′ to 5′ direction a-e.

A further aspect of the invention is a method to reduce the levels oftarget microRNA by contacting the target microRNA to an oligonucleotideas defined herein, wherein the oligonucleotide (i) is complementary tothe target microRNA sequence (ii) does not contain a nucleotide at the3′ end that corresponds to the first 5′ end nucleotide of the targetmicroRNA.

Duplex Stability and T_(m)

In one embodiment, the oligomer of the invention is capable of forming aduplex with a complementary single stranded RNA nucleic acid molecule(typically of about the same length of said single strandedoligonucleotide) with phosphodiester internucleoside linkages, whereinthe duplex has a T_(m) of between 30° C. and and 70° C. or 80° C., suchas between 30° C. and 60° C. to 70° C., or between 30° C. and 50° C. or60° C. In one embodiment the T_(m) is at least 40° C. T_(m) may bedetermined by determining the T_(m) of the oligomer and a complementaryRNA target in the following buffer conditions: 100 mM NaCl, 0.1 mM EDTA,10 mM Na-phosphate, pH 7.0 (see examples for a detailed protocol). Ahigh affinity analogue may be defined as an analogue which, when used inthe oligomer of the invention, results in an increase in the T_(m) ofthe oligomer as compared to an identical oligomer which has containsonly DNA bases.

Conjugates

In one embodiment, said oligomer is conjugated with one or morenon-nucleotide (or polynucleotide) compounds.

In the context the term “conjugate” is intended to indicate aheterogenous molecule formed by the covalent attachment (“conjugation”)of the oligomer as described herein to one or more non-nucleotide, ornon-polynucleotide moieties. Examples of non-nucleotide ornon-polynucleotide moieties include macromolecular agents such asproteins, fatty acid chains, sugar residues, glycoproteins, polymers, orcombinations thereof. Typically proteins may be antibodies for a targetprotein. Typical polymers may be polyethylene glycol.

Therefore, in various embodiments, the oligomer of the invention maycomprise both a polynucleotide region which typically consists of acontiguous sequence of nucleotides, and a further non-nucleotide region.When referring to the oligomer of the invention consisting of acontiguous nucleotide sequence, the compound may comprise non-nucleotidecomponents, such as a conjugate component.

In various embodiments of the invention the oligomeric compound islinked to ligands/conjugates, which may be used, e.g. to increase thecellular uptake of oligomeric compounds. WO2007/031091 provides suitableligands and conjugates, which are hereby incorporated by reference.

The invention also provides for a conjugate comprising the compoundaccording to the invention as herein described, and at least onenon-nucleotide or non-polynucleotide moiety covalently attached to saidcompound. Therefore, in various embodiments where the compound of theinvention consists of a specified nucleic acid or nucleotide sequence,as herein disclosed, the compound may also comprise at least onenon-nucleotide or non-polynucleotide moiety (e.g. not comprising one ormore nucleotides or nucleotide analogues) covalently attached to saidcompound.

Conjugation (to a conjugate moiety) may enhance the activity, cellulardistribution or cellular uptake of the oligomer of the invention. Suchmoieties include, but are not limited to, antibodies, polypeptides,lipid moieties such as a cholesterol moiety, cholic acid, a thioether,e.g. Hexyl-s-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipids, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or apolyethylene glycol chain, an adamantane acetic acid, a palmityl moiety,an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

The oligomers of the invention may also be conjugated to active drugsubstances, for example, aspirin, ibuprofen, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic.

In certain embodiments the conjugated moiety is a sterol, such ascholesterol.

In various embodiments, the conjugated moiety comprises or consists of apositively charged polymer, such as a positively charged peptides of,for example between 1-50, such as 2-20 such as 3-10 amino acid residuesin length, and/or polyalkylene oxide such as polyethylglycol(PEG) orpolypropylene glycol—see WO 2008/034123, hereby incorporated byreference. Suitably the positively charged polymer, such as apolyalkylene oxide may be attached to the oligomer of the invention viaa linker such as the releasable inker described in WO 2008/034123.

By way of example, the following conjugate moieties may be used in theconjugates of the invention:

Activated Oligomers

The term “activated oligomer,” as used herein, refers to an oligomer ofthe invention that is covalently linked (i.e., functionalized) to atleast one functional moiety that permits covalent linkage of theoligomer to one or more conjugated moieties, i.e., moieties that are notthemselves nucleic acids or monomers, to form the conjugates hereindescribed. Typically, a functional moiety will comprise a chemical groupthat is capable of covalently bonding to the oligomer via, e.g., a3′-hydroxyl group or the exocyclic NH₂ group of the adenine base, aspacer that is preferably hydrophilic and a terminal group that iscapable of binding to a conjugated moiety (e.g., an amino, sulfhydryl orhydroxyl group). In some embodiments, this terminal group is notprotected, e.g., is an NH₂ group. In other embodiments, the terminalgroup is protected, for example, by any suitable protecting group suchas those described in “Protective Groups in Organic Synthesis” byTheodora W Greene and Peter G M Wuts, 3rd edition (John Wiley & Sons,1999). Examples of suitable hydroxyl protecting groups include esterssuch as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, ortriphenylmethyl, and tetrahydropyranyl. Examples of suitable aminoprotecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl,triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groupssuch as trichloroacetyl or trifluoroacetyl. In some embodiments, thefunctional moiety is self-cleaving. In other embodiments, the functionalmoiety is biodegradable. See e.g., U.S. Pat. No. 7,087,229, which isincorporated by reference herein in its entirety.

In some embodiments, oligomers of the invention are functionalized atthe 5′ end in order to allow covalent attachment of the conjugatedmoiety to the 5′ end of the oligomer. In other embodiments, oligomers ofthe invention can be functionalized at the 3′ end. In still otherembodiments, oligomers of the invention can be functionalized along thebackbone or on the heterocyclic base moiety. In yet other embodiments,oligomers of the invention can be functionalized at more than oneposition independently selected from the 5′ end, the 3′ end, thebackbone and the base.

In some embodiments, activated oligomers of the invention aresynthesized by incorporating during the synthesis one or more monomersthat is covalently attached to a functional moiety. In otherembodiments, activated oligomers of the invention are synthesized withmonomers that have not been functionalized, and the oligomer isfunctionalized upon completion of synthesis. In some embodiments, theoligomers are functionalized with a hindered ester containing anaminoalkyl linker, wherein the alkyl portion has the formula (CH₂)_(w),wherein w is an integer ranging from 1 to 10, preferably about 6,wherein the alkyl portion of the alkylamino group can be straight chainor branched chain, and wherein the functional group is attached to theoligomer via an ester group (—O—C(O)—(CH₂)_(w)NH).

In other embodiments, the oligomers are functionalized with a hinderedester containing a (CH₂)_(w)-sulfhydryl (SH) linker, wherein w is aninteger ranging from 1 to 10, preferably about 6, wherein the alkylportion of the alkylamino group can be straight chain or branched chain,and wherein the functional group attached to the oligomer via an estergroup (—O—C(O)—(CH₂)_(w)SH).

In some embodiments, sulfhydryl-activated oligonucleotides areconjugated with polymer moieties such as polyethylene glycol or peptides(via formation of a disulfide bond).

Activated oligomers containing hindered esters as described above can besynthesized by any method known in the art, and in particular by methodsdisclosed in PCT Publication No. WO 2008/034122 and the examplestherein, which is incorporated herein by reference in its entirety.

In still other embodiments, the oligomers of the invention arefunctionalized by introducing sulfhydryl, amino or hydroxyl groups intothe oligomer by means of a functionalizing reagent substantially asdescribed in U.S. Pat. Nos. 4,962,029 and 4,914,210, i.e., asubstantially linear reagent having a phosphoramidite at one end linkedthrough a hydrophilic spacer chain to the opposing end which comprises aprotected or unprotected sulfhydryl, amino or hydroxyl group. Suchreagents primarily react with hydroxyl groups of the oligomer. In someembodiments, such activated oligomers have a functionalizing reagentcoupled to a 5′-hydroxyl group of the oligomer. In other embodiments,the activated oligomers have a functionalizing reagent coupled to a3′-hydroxyl group. In still other embodiments, the activated oligomersof the invention have a functionalizing reagent coupled to a hydroxylgroup on the backbone of the oligomer. In yet further embodiments, theoligomer of the invention is functionalized with more than one of thefunctionalizing reagents as described in U.S. Pat. Nos. 4,962,029 and4,914,210, incorporated herein by reference in their entirety. Methodsof synthesizing such functionalizing reagents and incorporating theminto monomers or oligomers are disclosed in U.S. Pat. Nos. 4,962,029 and4,914,210.

In some embodiments, the 5′-terminus of a solid-phase bound oligomer isfunctionalized with a dienyl phosphoramidite derivative, followed byconjugation of the deprotected oligomer with, e.g., an amino acid orpeptide via a Diels-Alder cycloaddition reaction.

In various embodiments, the incorporation of monomers containing2′-sugar modifications, such as a 2′-carbamate substituted sugar or a2′-(O-pentyl-N-phthalimido)-deoxyribose sugar into the oligomerfacilitates covalent attachment of conjugated moieties to the sugars ofthe oligomer. In other embodiments, an oligomer with an amino-containinglinker at the 2′-position of one or more monomers is prepared using areagent such as, for example,5′-dimethoxytrityl-2′-O-(e-phthalimidylaminopentyl)-2′-deoxyadenosine-3′-N,N-diisopropyl-cyanoethoxyphosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters,1991, 34, 7171.

In still further embodiments, the oligomers of the invention may haveamine-containing functional moieties on the nucleotide, including on theN6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or5 positions of cytosine. In various embodiments, such functionalizationmay be achieved by using a commercial reagent that is alreadyfunctionalized in the oligomer synthesis.

Some functional moieties are commercially available, for example,heterobifunctional and homobifunctional linking moieties are availablefrom the Pierce Co. (Rockford, Ill.). Other commercially availablelinking groups are 5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents,both available from Glen Research Corporation (Sterling, Va.).5′-Amino-Modifier C6 is also available from ABI (Applied BiosystemsInc., Foster City, Calif.) as Aminolink-2, and 3′-Amino-Modifier is alsoavailable from Clontech Laboratories Inc. (Palo Alto, Calif.).

Therapy and Pharmaceutical Compositions—Formulation and Administration

As explained initially, the oligonucleotides of the invention willconstitute suitable drugs with improved properties. The design of apotent and safe drug requires the fine-tuning of various parameters suchas affinity/specificity, stability in biological fluids, cellularuptake, mode of action, pharmacokinetic properties and toxicity.

Accordingly, in a further aspect the present invention relates to apharmaceutical composition comprising an oligonucleotide according tothe invention and a pharmaceutically acceptable diluent, carrier oradjuvant. Preferably said carrier is saline or buffered saline.

In a still further aspect the present invention relates to anoligonucleotide according to the present invention for use as amedicament.

As will be understood, dosing is dependent on severity andresponsiveness of the disease state to be treated, and the course oftreatment lasting from several days to several months, or until a cureis effected or a diminution of the disease state is achieved. Optimaldosing schedules can be calculated from measurements of drugaccumulation in the body of the patient. Optimum dosages may varydepending on the relative potency of individual oligonucleotides.Generally it can be estimated based on EC50s found to be effective in invitro and in vivo animal models. In general, dosage is from 0.01 μg to 1g per kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 10 years or by continuousinfusion for hours up to several months. The repetition rates for dosingcan be estimated based on measured residence times and concentrations ofthe drug in bodily fluids or tissues. Following successful treatment, itmay be desirable to have the patient undergo maintenance therapy toprevent the recurrence of the disease state.

As indicated above, the invention also relates to a pharmaceuticalcomposition, which comprises at least one oligonucleotide of theinvention as an active ingredient. It should be understood that thepharmaceutical composition according to the invention optionallycomprises a pharmaceutical carrier, and that the pharmaceuticalcomposition optionally comprises further compounds, such aschemotherapeutic compounds, anti-inflammatory compounds, antiviralcompounds and/or immuno-modulating compounds.

The oligonucleotides of the invention can be used “as is” or in form ofa variety of pharmaceutically acceptable salts. As used herein, the term“pharmaceutically acceptable salts” refers to salts that retain thedesired biological activity of the herein-identified oligonucleotidesand exhibit minimal undesired toxicological effects. Non-limitingexamples of such salts can be formed with organic amino acid and baseaddition salts formed with metal cations such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium,potassium, and the like, or with a cation formed from ammonia,N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, orethylenediamine.

In one embodiment of the invention, the oligonucleotide may be in theform of a pro-drug. Oligonucleotides are by virtue negatively chargedions. Due to the lipophilic nature of cell membranes the cellular uptakeof oligonucleotides are reduced compared to neutral or lipophilicequivalents. This polarity “hindrance” can be avoided by using thepro-drug approach (see e.g. Crooke, R. M. (1998) in Crooke, S. T.Antisense research and Application. Springer-Verlag, Berlin, Germany,vol. 131, pp. 103-140).

Pharmaceutically acceptable binding agents and adjuvants may comprisepart of the formulated drug.

Examples of delivery methods for delivery of the therapeutic agentsdescribed herein, as well as details of pharmaceutical formulations,salts, may are well described elsewhere for example in U.S. provisionalapplication 60/838,710 and 60/788,995, which are hereby incorporated byreference, and Danish applications, PA 2006 00615 which is also herebyincorporated by reference.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Delivery ofdrug to tumour tissue may be enhanced by carrier-mediated deliveryincluding, but not limited to, cationic liposomes, cyclodextrins,porphyrin derivatives, branched chain dendrimers, polyethyleniminepolymers, nanoparticles and microspheres (Dass C R. J Pharm Pharmacol2002; 54(1):3-27). The pharmaceutical formulations of the presentinvention, which may conveniently be presented in unit dosage form, maybe prepared according to conventional techniques well known in thepharmaceutical industry. Such techniques include the step of bringinginto association the active ingredients with the pharmaceuticalcarrier(s) or excipient(s). In general the formulations are prepared byuniformly and intimately bringing into association the activeingredients with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product. The compositions ofthe present invention may be formulated into any of many possible dosageforms such as, but not limited to, tablets, capsules, gel capsules,liquid syrups, soft gels and suppositories. The compositions of thepresent invention may also be formulated as suspensions in aqueous,non-aqueous or mixed media. Aqueous suspensions may further containsubstances which increase the viscosity of the suspension including, forexample, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension may also contain stabilizers. The compounds of the inventionmay also be conjugated to active drug substances, for example, aspirin,ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or anantibiotic.

In another embodiment, compositions of the invention may contain one ormore oligonucleotide compounds, targeted to a first microRNA and one ormore additional oligonucleotide compounds targeted to a second microRNAtarget. Two or more combined compounds may be used together orsequentially.

The compounds disclosed herein are useful for a number of therapeuticapplications as indicated above. In general, therapeutic methods of theinvention include administration of a therapeutically effective amountof an oligonucleotide to a mammal, particularly a human. In a certainembodiment, the present invention provides pharmaceutical compositionscontaining (a) one or more compounds of the invention, and (b) one ormore chemotherapeutic agents. When used with the compounds of theinvention, such chemotherapeutic agents may be used individually,sequentially, or in combination with one or more other suchchemotherapeutic agents or in combination with radiotherapy. Allchemotherapeutic agents known to a person skilled in the art are hereincorporated as combination treatments with compound according to theinvention. Other active agents, such as anti-inflammatory drugs,including but not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, antiviral drugs, and immuno-modulating drugs may alsobe combined in compositions of the invention. Two or more combinedcompounds may be used together or sequentially.

Examples of therapeutic indications which may be treated by thepharmaceutical compositions of the invention:

microRNA Possible medical indications miR-1 Cardiac arythmia miR-21Glioblastoma, breast cancer, hepatocellular carcinoma, colorectalcancer, sensitization of gliomas to cytotoxic drugs, cardiac hypertrophymiR-21, miR-200b Response to chemotherapy and regulation of and miR-141cholangiocarcinoma growth miR-122 hypercholesterolemia, hepatitis Cinfection, hemochromatosis miR-19b lymphoma and other tumour typesmiR-26a Osteoblast differentiation of human stem cells miR-155 lymphoma,pancreatic tumor development, breast and lung cancer miR-203 PsoriasismiR-375 diabetes, metabolic disorders, glucose- induced insulinsecretion from pancreatic endocrine cells miR-181 myoblastdifferentiation, auto immune disorders miR-10b Breast cancer cellinvasion and metastasis miR-125b-1 Breast, lung, ovarian and cervicalcancer miR-221 and 222 Prostate carcinoma, human thyroid papillary car,human hepatocellular carcinoma miRNA-372 and -373 testicular germ celltumors. miR-142 B-cell leukemia miR-17-19b B-cell lymphomas, lungcancer, hepatocellular cluster carcinoma

Tumor suppressor gene tropomysin 1 (TPM1) mRNA has been indicated as atarget of miR-21. Myotrophin (mtpn) mRNA has been indicated as a targetof miR 375.

In an even further aspect, the present invention relates to the use ofan oligonucleotide according to the invention for the manufacture of amedicament for the treatment of a disease selected from the groupconsisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia;cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes,metabolic disorders; myoblast differentiation; immune disorders.

The invention further refers to oligonucleotides according to theinvention for the use in the treatment of from a disease selected fromthe group consisting of: atherosclerosis, hypercholesterolemia andhyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lungcancer; diabetes, metabolic disorders; myoblast differentiation; immunedisorders.

The invention provides for a method of treating a subject suffering froma disease or condition selected from from the group consisting of:atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer,glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolicdisorders; myoblast differentiation; immune disorders, the methodcomprising the step of administering an oligonucleotide orpharmaceutical composition of the invention to the subject in needthereof.

The invention further provides for a kit comprising a pharmaceuticalcomposition according to the invention, and a second independent activeingredient that is an inhibitor of the VLDL assembly pathway, such as anApoB inhibitor, or an MTP inhibitor.

Cancer

In an even further aspect, the present invention relates to the use ofan oligonucleotide according to the invention for the manufacture of amedicament for the treatment of cancer. In another aspect, the presentinvention concerns a method for treatment of, or prophylaxis against,cancer, said method comprising administering an oligonucleotide of theinvention or a pharmaceutical composition of the invention to a patientin need thereof.

Such cancers may include lymphoreticular neoplasia, lymphoblasticleukemia, brain tumors, gastric tumors, plasmacytomas, multiple myeloma,leukemia, connective tissue tumors, lymphomas, and solid tumors.

In the use of a compound of the invention for the manufacture of amedicament for the treatment of cancer, said cancer may suitably be inthe form of a solid tumor. Analogously, in the method for treatingcancer disclosed herein said cancer may suitably be in the form of asolid tumor.

Furthermore, said cancer is also suitably a carcinoma. The carcinoma istypically selected from the group consisting of malignant melanoma,basal cell carcinoma, ovarian carcinoma, breast carcinoma, non-smallcell lung cancer, renal cell carcinoma, bladder carcinoma, recurrentsuperficial bladder cancer, stomach carcinoma, prostatic carcinoma,pancreatic carcinoma, lung carcinoma, cervical carcinoma, cervicaldysplasia, laryngeal papillomatosis, colon carcinoma, colorectalcarcinoma and carcinoid tumors. More typically, said carcinoma isselected from the group consisting of malignant melanoma, non-small celllung cancer, breast carcinoma, colon carcinoma and renal cell carcinoma.The malignant melanoma is typically selected from the group consistingof superficial spreading melanoma, nodular melanoma, lentigo malignamelanoma, acral melagnoma, amelanotic melanoma and desmoplasticmelanoma.

Alternatively, the cancer may suitably be a sarcoma. The sarcoma istypically in the form selected from the group consisting ofosteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibroushistiocytoma, fibrosarcoma and Kaposi's sarcoma.

Alternatively, the cancer may suitably be a glioma.

A further embodiment is directed to the use of an oligonucleotideaccording to the invention for the manufacture of a medicament for thetreatment of cancer, wherein said medicament further comprises achemotherapeutic agent selected from the group consisting ofadrenocorticosteroids, such as prednisone, dexamethasone or decadron;altretamine (hexalen, hexamethylmelamine (HMM)); amifostine (ethyol);aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole (anmidex);androgens, such as testosterone; asparaginase (elspar); bacilluscalmette-gurin; bicalutamide (casodex); bleomycin (blenoxane); busulfan(myleran); carboplatin (paraplatin); carmustine (BCNU, BiCNU);chlorambucil (leukeran); chlorodeoxyadenosine (2-CDA, cladribine,leustatin); cisplatin (platinol); cytosine arabinoside (cytarabine);dacarbazine (DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin(cerubidine); docetaxel (taxotere); doxorubicin (adriomycin);epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol(DES); etopside (VP-16, VePesid, etopophos); fludarabine (fludara);flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU);gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab);hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2(proleukin, aldesleukin); interferon alpha (intron A, roferon A);irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole);lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard);melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate(mexate); mitomycin-C (mutamucin); mitoxantrone (novantrone); octreotide(sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin(mithramycin, mithracin); prorocarbazine (matulane); streptozocin;tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon, VM-26);thiotepa; topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoicacid); vinblastine (valban); vincristine (oncovin) and vinorelbine(navelbine). Suitably, the further chemotherapeutic agent is selectedfrom taxanes such as Taxol, Paclitaxel or Docetaxel.

Similarly, the invention is further directed to the use of anoligonucleotide according to the invention for the manufacture of amedicament for the treatment of cancer, wherein said treatment furthercomprises the administration of a further chemotherapeutic agentselected from the group consisting of adrenocorticosteroids, such asprednisone, dexamethasone or decadron; altretamine (hexalen,hexamethylmelamine (HMM)); amifostine (ethyol); aminoglutethimide(cytadren); amsacrine (M-AMSA); anastrozole (arimidex); androgens, suchas testosterone; asparaginase (elspar); bacillus calmette-gurin;bicalutamide (casodex); bleomycin (blenoxane); busulfan (myleran);carboplatin (paraplatin); carmustine (BCNU, BiCNU); chlorambucil(leukeran); chlorodeoxyadenosine (2-CDA, cladribine, leustatin);cisplatin (platinol); cytosine arabinoside (cytarabine); dacarbazine(DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin(cerubidine); docetaxel (taxotere); doxorubicin (adriomycin);epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol(DES); etopside (VP-16, VePesid, etopophos); fludarabine (fludara);flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU);gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab);hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2(proleukin, aldesleukin); interferon alpha (intron A, roferon A);irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole);lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard);melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate(mexate); mitomycin-C (mutamucin); mitoxantrone (novantrone); octreotide(sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin(mithramycin, mithracin); prorocarbazine (matulane); streptozocin;tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon, VM-26);thiotepa; topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoicacid); vinblastine (valban); vincristine (oncovin) and vinorelbine(navelbine). Suitably, said treatment further comprises theadministration of a further chemotherapeutic agent selected fromtaxanes, such as Taxol, Paclitaxel or Docetaxel.

Alternatively stated, the invention is furthermore directed to a methodfor treating cancer, said method comprising administering anoligonucleotide of the invention or a pharmaceutical compositionaccording to the invention to a patient in need thereof and furthercomprising the administration of a further chemotherapeutic agent. Saidfurther administration may be such that the further chemotherapeuticagent is conjugated to the compound of the invention, is present in thepharmaceutical composition, or is administered in a separateformulation.

Infectious Diseases

It is contemplated that the compounds of the invention may be broadlyapplicable to a broad range of infectious diseases, such as diphtheria,tetanus, pertussis, polio, hepatitis B, hepatitis C, hemophilusinfluenza, measles, mumps, and rubella.

Hsa-miR122 is indicated in hepatitis C infection and as sucholigonucleotides according to the invention which target miR-122 may beused to treat Hepatitus C infection.

Accordingly, in yet another aspect the present invention relates the useof an oligonucleotide according to the invention for the manufacture ofa medicament for the treatment of an infectious disease, as well as to amethod for treating an infectious disease, said method comprisingadministering an oligonucleotide according to the invention or apharmaceutical composition according to the invention to a patient inneed thereof.

In a preferred embodiment, the invention provides for a combinationtreatment providing an anti miR-122 oligomer in combination with aninhibitor of VLDL assembly, such as an inhibitor of apoB, or of MTP.

Inflammatory Diseases

The inflammatory response is an essential mechanism of defense of theorganism against the attack of infectious agents, and it is alsoimplicated in the pathogenesis of many acute and chronic diseases,including autoimmune disorders. In spite of being needed to fightpathogens, the effects of an inflammatory burst can be devastating. Itis therefore often necessary to restrict the symptomatology ofinflammation with the use of anti-inflammatory drugs. Inflammation is acomplex process normally triggered by tissue injury that includesactivation of a large array of enzymes, the increase in vascularpermeability and extravasation of blood fluids, cell migration andrelease of chemical mediators, all aimed to both destroy and repair theinjured tissue.

In yet another aspect, the present invention relates to the use of anoligonucleotide according to the invention for the manufacture of amedicament for the treatment of an inflammatory disease, as well as to amethod for treating an inflammatory disease, said method comprisingadministering an oligonucleotide according to the invention or apharmaceutical composition according to the invention to a patient inneed thereof.

In one preferred embodiment of the invention, the inflammatory diseaseis a rheumatic disease and/or a connective tissue diseases, such asrheumatoid arthritis, systemic lupus erythematous (SLE) or Lupus,scleroderma, polymyositis, inflammatory bowel disease, dermatomyositis,ulcerative colitis, Crohn's disease, vasculitis, psoriatic arthritis,exfoliative psoriatic dermatitis, pemphigus vulgaris and Sjorgren'ssyndrome, in particular inflammatory bowel disease and Crohn's disease.

Alternatively, the inflammatory disease may be a non-rheumaticinflammation, like bursitis, synovitis, capsulitis, tendinitis and/orother inflammatory lesions of traumatic and/or sportive origin.

Metabolic Diseases

A metabolic disease is a disorder caused by the accumulation ofchemicals produced naturally in the body. These diseases are usuallyserious, some even life threatening. Others may slow physicaldevelopment or cause mental retardation. Most infants with thesedisorders, at first, show no obvious signs of disease. Proper screeningat birth can often discover these problems. With early diagnosis andtreatment, metabolic diseases can often be managed effectively.

In yet another aspect, the present invention relates to the use of anoligonucleotide according to the invention or a conjugate thereof forthe manufacture of a medicament for the treatment of a metabolicdisease, as well as to a method for treating a metabolic disease, saidmethod comprising administering an oligonucleotide according to theinvention or a conjugate thereof, or a pharmaceutical compositionaccording to the invention to a patient in need thereof.

In one preferred embodiment of the invention, the metabolic disease isselected from the group consisting of Amyloidosis, Biotinidase, OMIM(Online Mendelian Inheritance in Man), Crigler Najjar Syndrome,Diabetes, Fabry Support & Information Group, Fatty acid OxidationDisorders, Galactosemia, Glucose-6-Phosphate Dehydrogenase (G6PD)deficiency, Glutaric aciduria, International Organization of GlutaricAcidemia, Glutaric Acidemia Type I, Glutaric Acidemia, Type II, GlutaricAcidemia Type I, Glutaric Acidemia Type-II, F-HYPDRR—FamilialHypophosphatemia, Vitamin D Resistant Rickets, Krabbe Disease, Longchain 3 hydroxyacyl CoA dehydrogenase deficiency (LCHAD), MannosidosisGroup, Maple Syrup Urine Disease, Mitochondrial disorders,Mucopolysaccharidosis Syndromes: Niemann Pick, Organic acidemias, PKU,Pompe disease, Porphyria, Metabolic Syndrome, Hyperlipidemia andinherited lipid disorders, Trimethylaminuria: the fish malodor syndrome,and Urea cycle disorders.

Liver Disorders

In yet another aspect, the present invention relates to the use of anoligonucleotide according to the invention or a conjugate thereof forthe manufacture of a medicament for the treatment of a liver disorder,as well as to a method for treating a liver disorder, said methodcomprising administering an oligonucleotide according to the inventionor a conjugate thereof, or a pharmaceutical composition according to theinvention to a patient in need thereof.

In one preferred embodiment of the invention, the liver disorder isselected from the group consisting of Biliary Atresia, AlagilleSyndrome, Alpha-1 Antitrypsin, Tyrosinemia, Neonatal Hepatitis, andWilson Disease.

Other Uses

The oligonucleotides of the present invention can be utilized for asresearch reagents for diagnostics, therapeutics and prophylaxis. Inresearch, the oligonucleotide may be used to specifically inhibit thesynthesis of target genes in cells and experimental animals therebyfacilitating functional analysis of the target or an appraisal of itsusefulness as a target for therapeutic intervention. In diagnostics theoligonucleotides may be used to detect and quantitate target expressionin cell and tissues by Northern blotting, in-situ hybridisation orsimilar techniques. For therapeutics, an animal or a human, suspected ofhaving a disease or disorder, which can be treated by modulating theexpression of target is treated by administering the oligonucleotidecompounds in accordance with this invention. Further provided aremethods of treating an animal particular mouse and rat and treating ahuman, suspected of having or being prone to a disease or condition,associated with expression of target by administering a therapeuticallyor prophylactically effective amount of one or more of theoligonucleotide compounds or compositions of the invention.

Therapeutic Use of Oligonucleotdes Targeting miR-122a

We have demonstrated that a LNA-antimiR, targeting miR-122a reducesplasma cholesterol levels. Therefore, another aspect of the invention isuse of the above described oligonucleotides targeting miR-122a asmedicine.

Still another aspect of the invention is use of the above describedoligonucleotides targeting miR-122a for the preparation of a medicamentfor treatment of increased plasma cholesterol levels (orhypercholesterolemia and related disorders). The skilled man willappreciate that increased plasma cholesterol levels is undesireable asit increases the risk of various conditions, e.g. atherosclerosis.

Still another aspect of the invention is use of the above describedoligonucleotides targeting miR-122a for upregulating the mRNA levels ofNrdg3, Aldo A, Bckdk or CD320.

EMBODIMENTS

The following embodiments of the present invention may be used incombination with the other embodiments described herein.

1. A pharmaceutical composition comprising an oligomer of between 6-12nucleotides in length, wherein said oligomer comprises a contiguousnucleotide sequence of a total of between 6-12 nucleotides, such as 6,7, 8, 9, 10, 11 or 12 nucleotide units, wherein at least 50% of thenucleobase units of the oligomer are high affinity nucleotide analogueunits, and a pharmaceutically acceptable diluent, carrier, salt oradjuvant.2. The pharmaceutical composition according to embodiment 1, wherein thecontiguous nucleotide sequence is complementary to a correspondingregion of a mammalian, human or viral microRNA (miRNA) sequence.3. The pharmaceutical composition according to embodiment 2, wherein thecontiguous nucleotide sequence is complementary to a correspondingregion of a miRNA sequence selected from the group of miRNAs listed inany one of tables 3, 4 or 5.4. The pharmaceutical composition according to embodiment 2 or 3,wherein the contiguous nucleotide sequence consists of or comprises asequence which is complementary to the seed sequence of said microRNA.5. The pharmaceutical composition according to any one of embodiments2-4, wherein the contiguous nucleotide sequence consists of or comprisesa sequence selected from any one of the sequences listed in table 3 or4.6. The pharmaceutical composition according to embodiment 4 or 5,wherein the 3′ nucleobase of the seedmer forms the 3′ most nucleobase ofthe contiguous nucleotide sequence, wherein the contiguous nucleotidesequence may, optionally, comprise one or two further 5′ nucleobases.7. The pharmaceutical composition according to any one of embodiments1-6, wherein said contiguous nucleotide sequence does not comprise anucleotide which corresponds to the first nucleotide present in themicro RNA sequence counted from the 5′ end.8. The pharmaceutical composition according to any one of embodiments1-7, wherein the contiguous nucleotide sequence is complementary to acorresponding nucleotide sequence present in a miRNA selected from thoseshown in table 3 or 4 or 5.9. The pharmaceutical composition according to embodiment 8, whereinsaid miRNA is selected from the group consisting of miR-1, miR-10b,miR-17-3p, miR-18, miR-19a, miR-19b, miR-20, miR-21, miR-34a, miR-93,miR-106a, miR-106b, miR-122, miR-133, miR-134, miR-138, miR-155,miR-192, miR-194, miR-221, miR-222, and miR-375.10. The pharmaceutical composition according to any one of embodiments1-9, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or all ofthe nucleobase units of the contiguous nucleotide sequence arenucleotide analogue units.11. The pharmaceutical composition according to embodiment 10, whereinthe nucleotide analogue units are selected from the group consisting of2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNAunit, LNA unit, PNA unit, HNA unit, INA unit, and a 2′MOE RNA unit.12. The pharmaceutical composition according to embodiment 10 or 11,wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or all of thenucleobase units of the contiguous nucleotide sequence are LockedNucleic Acid (LNA) nucleobase units.13. The pharmaceutical composition according to embodiment 12, whereinall of the nucleobase units of the contiguous nucleotide sequence areLNA nucleobase units.14. The pharmaceutical composition according to any one of embodiments1-13, wherein the contiguous nucleotide sequence comprises or consistsof 7, 8, 9 or 10, preferably contiguous, LNA nucleobase units.15. The pharmaceutical composition according to any one of embodiments1-14, wherein the oligomer consist of 7, 8, 9 or 10 contiguousnucleobase units and wherein at least 7 nucleobase units are nucleotideanalogue units.16. The pharmaceutical composition according to embodiment 15, whereinthe nucleotide analogue units are Locked Nucleic Acid (LNA) nucleobaseunits.17. The pharmaceutical composition according to embodiment 15, whereinthe nucleotide analogue units in the molecule consists of a mixture ofat least 50% LNA units and up to 50% other nucleotide analogue units.18. The pharmaceutical composition according to any one of embodiments1-17, wherein at least 75%, such as 80% or 85% or 90% or 95% or all ofthe internucleoside linkages present between the nucleobase units of thecontiguous nucleotide sequence are phosphorothioate internucleosidelinkages.19. The pharmaceutical composition according to any one of embodiments1-18, wherein said oligomer is conjugated with one or morenon-nucleobase compounds.20. The pharmaceutical composition according to any one of embodiments1-19, wherein the contiguous nucleotide sequence is complementary to thecorresponding sequence of at least two miRNA sequences such as 2, 3, 4,5, 6, 7, 8, 9, or 10 miRNA sequences.21. The pharmaceutical composition according to any one of embodiments1-20, wherein the contiguous nucleotide sequence consists or comprisesof a sequence which is complementary to the sequence of at least twomiRNA seed region sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNAseed region sequences.22. The pharmaceutical composition according to any one of embodiments20 or 21, wherein the contiguous nucleotide sequence is complementary tothe corresponding region of both miR-221 and miR-222.23. The pharmaceutical composition according to embodiment 22, whereinthe contiguous nucleotide sequence consists or comprises of a sequencethat is complementary to 5′GCUACAU3′.24. The pharmaceutical composition according to any one of embodiments1-23, wherein the oligomer is constituted as a prodrug.25. The pharmaceutical composition according to any one of embodiments1-24, wherein the contiguous nucleotide sequence is complementary to acorresponding region of has-miR-122.26. The pharmaceutical composition according to embodiment 25, for usein the treatment of a medical disorder or disease selected from thegroup consisting of: hepatitis C virus infection andhypercholesterolemia and related disorders.27. The pharmaceutical composition according to embodiment 25 or 26,wherein the composition further comprises a second independent activeingredient that is an inhibitor of the VLDL assembly pathway, such as anApoB inhibitor, or an MTP inhibitor.28. A kit comprising a pharmaceutical composition according toembodiment 25 or 26, and a second independent active ingredient that isan inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor, oran MTP inhibitor.29. A method for the treatment of a disease or medical disorderassociated with the presence or overexpression of a microRNA, comprisingthe step of administering a the pharmaceutical composition) according toany one of embodiments 1-28 to a patient who is suffering from, or islikely to siffer from said disease or medical disorder.30. An oligomer, as defined according to anyone of embodiments 1-25.31. A conjugate comprising the oligomer according to embodiment 30, andat least one non-nucleobase compounds.32. The use of an oligomer or a conjugate as defined in any one ofembodiments 30-31, for the manufacture of a medicament for the treatmentof a disease or medical disorder associated with the presence orover-expression of the microRNA.33. A method for reducing the amount, or effective amount, of a miRNA ina cell, comprising administering an oligomer, a conjugate or apharmaceutical composition, according to any one of the preceedingembodiments to the cell which is expressing said miRNA so as to reducethe amount, or effective amount of the miRNA in the cell.34. A method for de-repression of a mRNA whose expression is repressedby a miRNA in a cell comprising administering an oligomer, a conjugateor a pharmaceutical composition, according to any one of the preceedingembodiments to the cell to the cell which expressed both said mRNA andsaid miRNA, in order to de-repress the expression of the mRNA.References: Details of the reference are provided in the prioritydocuments.

EXAMPLES

LNA Monomer and oligonucleotide synthesis were performed using themethodology referred to in Examples 1 and 2 of WO2007/112754. Thestability of LNA oligonucletides in human or rat plasma is performedusing the methodology referred to in Example 4 of WO2007/112754. Thetreatment of in vitro cells with LNA anti-miR antisense oligonucleotide(targeting miR-122) is performed using the methodology referred to inExample 6 of WO2007/112754. The analysis of Oligonucleotide Inhibitionof miR expression by microRNA specific quantitative PCR in both an invitro and in vivo model is performed using the methodology referred toin Example 7 of WO2007/112754. The assessment of LNA antimir knock-downspecificity using miRNA microarray expression profiling is performedusing the methodology referred to in Example 8 of WO2007/112754. Thedetection of microRNAs by in situ hybridization is performed using themethodology referred to in Example 9 of WO2007/112754. The Isolation andanalysis of mRNA expression (total RNA isolation and cDNA synthesis formRNA analysis) in both an in vitro and in vivo model is performed usingthe methodology referred to in Example 10 of WO2007/112754. In vivoExperiments using Oligomers of the invention targeting microRNA-122. andsubsequent analysis are performed using the methods disclosed inExamples 11-27 of WO2007/112754. The above mentioned examples ofWO2007/112754 are hereby specifically incorporated by reference.

Example 1: Design of the LNA antimiR Oligonucleotides and MeltingTemperatures

TABLE 2 Oligomers used in the examples and figures. The SEQ#is an identifier usedthroughout the examples and figures-the SEQ ID NO which is used in the sequencelisting is also provided. Example/Figure SEQ ID SEQ # NOCompound Sequence Comment #3204   1 TcAGtCTGaTaAgCT #3205   2 GATAAGCT#3206   3 TcAcAATtaGCAtTA #3207   4 TAGCATTA #4   5 CcAttGTcaCaCtCC#3208   6 CACACTCC #3209   7 TAAGCT #3210   8 ATAAGCT #3211   9TGATAAGCT #3212  10 CTGATAAGCT #3213  11 GTCTGATAAGCT #2114  12CAGTCTGATAAGCT #3215  13 TCTGATAA #3216  14 ATCAGTCT #3217  15 TCAACATC#3218/#3230  16 GGTAAACT Underline = mismatch #3219  17 CGTAATGAUnderline = mismatch #3220  18 TCAgtctgataaGCTa 5′fluorescent label (FAM) #3221  19 AGCACTTT #3222  20 ATTTGCAC #3223  21AgCagACaaTgTaGC 5′ fluorescent label (FAM) #3224  22 GtAgcCAgaTgTaGC 5′fluorescent label (FAM) #3225  23 ATGTAGC #3226  24 ACaAcCTacTaCcTC#3227  25 ACTACCTC #3228  26 CaCtgTCagCaCtTT #3229  27 TgCatAGatTtGcAC#3231  28 GTAGACT #3232  29 TACCTC #3233  30 CTACCTC #3234  31 TNCTACCTCN = universal base. #3235  32 TNCTACCTC N = universal base. #3236  33GCaAcCTacTaCcTC #3237  34 ACaAcCTccTaCcTC #3238  35 ACaAaCTacTaCcTC#3239  36 CTACCTC #3240  37 CTAACTC #3241  38 TTAGCATTA #3242  39CGATTAGCATTA #3243 977 CACGATTAGCATTA #3244 978 GCATTA #3245 979 AGCATTA#3246 980 ATTAGCATTA Capital and lower case letters denote LNA and DNA,respectively. LNA cytosines are preferably methylcytosine/5′methyl-cytosine* All internucleoside linkages are preferablyphosphorothioate* All LNA may, for example, be beta-D-oxy LNA* *Used inthe specific examples.

Example 2: In Vitro Model: Cell Culture

The effect of LNA oligonucleotides on target nucleic acid expression(amount) can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. Target can beexpressed endogenously or by transient or stable transfection of anucleic acid encoding said nucleic acid.

The expression level of target nucleic acid can be routinely determinedusing, for example, Northern blot analysis (including microRNAnorthern), Quantitative PCR (including microRNA qPCR), Ribonucleaseprotection assays. The following cell types are provided forillustrative purposes, but other cell types can be routinely used,provided that the target is expressed in the cell type chosen.

Cells were cultured in the appropriate medium as described below andmaintained at 37° C. at 95-98% humidity and 5% CO₂. Cells were routinelypassaged 2-3 times weekly.

15PC3: The human prostate cancer cell line 15PC3 was kindly donated byDr. F. Baas, Neurozintuigen Laboratory, AMC, The Netherlands and wascultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+GlutamaxI+gentamicin.

PC3 The human prostate cancer cell line PC3 was purchased from ATCC andwas cultured in F12 Coon's with glutamine (Gibco)+10% FBS+gentamicin.

518A2: The human melanoma cancer cell line 518A2 was kindly donated byDr. B. Jansen, Section of experimental Oncology, Molecular Pharmacology,Department of Clinical Pharmacology, University of Vienna and wascultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+GlutamaxI+gentamicin.HeLa: The cervical carcinoma cell line HeLa was cultured in MEM (Sigma)containing 10% fetal bovine serum gentamicin at 37° C., 95% humidity and5% CO₂.MPC-11: The murine multiple myeloma cell line MPC-11 was purchased fromATCC and maintained in DMEM with 4 mM Glutamax+10% Horse Serum.DU-145: The human prostate cancer cell line DU-145 was purchased fromATCC and maintained in RPMI with Glutamax+10% FBS.RCC-4+/−VHL: The human renal cancer cell line RCC4 stably transfectedwith plasmid expressing VHL or empty plasmid was purchased from ECACCand maintained according to manufacturers instructions.786-0: The human renal cell carcinoma cell line 786-0 was purchased fromATCC and maintained according to manufacturers instructionsHUVEC: The human umbilical vein endothelial cell line HUVEC waspurchased from Camcrex and maintained in EGM-2 medium.K562: The human chronic myelogenous leukaemia cell line K562 waspurchased from ECACC and maintained in RPMI with Glutamax+10% FBS.U87MG: The human glioblastoma cell line U87MG was purchased from ATCCand maintained according to the manufacturers instructions.B16: The murine melanoma cell line B16 was purchased from ATCC andmaintained according to the manufacturers instructions.LNCap: The human prostate cancer cell line LNCap was purchased from ATCCand maintained in RPMI with Glutamax+10% FBSHuh-7: Human liver, epithelial like cultivated in Eagles MEM with 10%FBS, 2 mM Glutamax I, 1× non-essential amino acids, Gentamicin 25 μg/mlL428: (Deutsche Sammlung für Mikroorganismen (DSM, Braunschwieg,Germany)): Human B cell lymphoma maintained in RPMI 1640 supplementedwith 10% FCS, L-glutamine and antibiotics.L1236: (Deutsche Sammlung für Mikroorganismen (DSM, Braunschwieg,Germany)): Human B cell lymphoma maintained in RPMI 1640 supplementedwith 10% FCS, L-glutamine and antibiotics.

Example 3: Design of a LNA antimiR Library for all Human microRNASequences in miRBase microRNA Database

The miRBase version used was version 12, as reported in Griffiths-Jones,S., Grocock, R. J., van Dongen, S., Bateman, A., Enright, A. J. 2006.miRBase: microRNA sequences, targets and gene nomenclature. NucleicAcids Res. 34: D140-4, and available viahttp://microma.sanger.ac.uk/sequences/index.shtml.

Table 1 shows 7, 8 and 9mer nucleotide sequences comprising the seedmersequence of micro RNA's according to the miRBase micro RNA database. Theseedmer sequence comprises the reverse complement of the microRNA seedregion. In some embodiments the oligomer of the invention has acontiguous nucleotide sequence selected from the 7mer, 8mer or 9mersequences. With respect to the 7mer, 8mer and 9mer sequences, in someembodiments, all the internucleoside linkages are phosphorothioate. The7mer, 8mer and 9mer nucleotide sequences may consist of sequence ofnucleotide analogues as described herein, such as LNA nucleotideanalogues. LNA cytosines may be methyl-cytosine (5′methyl-cytosine). Insome embodiments, the LNA is beta-D-oxy-LNA.

Table 3 provides a list of microRNAs grouped into those which can betargeted by the same seedmer oligomers, such as the 7, 8 or 9mersprovided herein (see table 1).

TABLE 3 hsa-let-7a*, hsa-let-7f-1* hsa-let-7a, hsa-let-7b, hsa-let-7c,hsa-let-7d, hsa-let-7f, hsa-miR-98, hsa-let-7g, hsa-let-7i hsa-miR-1,hsa-miR-206 hsa-miR-103, hsa-miR-107 hsa-miR-10a, hsa-miR-10bhsa-miR-125b, hsa-miR-125a-5p hsa-miR-129*, hsa-miR-129-3p hsa-miR-130a,hsa-miR-301a, hsa-miR-130b, hsa-miR-454, hsa-miR-301b hsa-miR-133a,hsa-miR-133b hsa-miR-135a, hsa-miR-135b hsa-miR-141, hsa-miR-200ahsa-miR-146a, hsa-miR-146b-5p hsa-miR-152, hsa-miR-148b hsa-miR-154*,hsa-miR-487a hsa-miR-15a, hsa-miR-16, hsa-miR-15b, hsa-miR-195,hsa-miR-497 hsa-miR-17, hsa-miR-20a, hsa-miR-93, hsa-miR-106a,hsa-miR-106b, hsa-miR-20b, hsa-miR-526b* hsa-miR-181a, hsa-miR-181chsa-miR-181b, hsa-miR-181d hsa-miR-18a, hsa-miR-18b hsa-miR-190,hsa-miR-190b hsa-miR-192, hsa-miR-215 hsa-miR-196a, hsa-miR-196bhsa-miR-199a-3p, hsa-miR-199b-3p hsa-miR-199a-5p, hsa-miR-199b-5phsa-miR-19a*, hsa-miR-19b-1*, hsa-miR-19b-2* hsa-miR-19a, hsa-miR-19bhsa-miR-200b, hsa-miR-200c hsa-miR-204, hsa-miR-211 hsa-miR-208a,hsa-miR-208b hsa-miR-212, hsa-miR-132 hsa-miR-23a*, hsa-miR-23b*hsa-miR-23a, hsa-miR-23b, hsa-miR-130a* hsa-miR-24-1*, hsa-miR-24-2*hsa-miR-25, hsa-miR-92a, hsa-miR-367, hsa-miR-92b hsa-miR-26a,hsa-miR-26b hsa-miR-26a-1*, hsa-miR-26a-2* hsa-miR-27a, hsa-miR-27bhsa-miR-29a, hsa-miR-29b, hsa-miR-29c hsa-miR-302a, hsa-miR-302b,hsa-miR-302c, hsa-miR-302d, hsa-miR-373, hsa-miR-520e, hsa-miR-520a-3p,hsa-miR-520b, hsa-miR-520c-3p, hsa-miR-520d-3p hsa-miR-302b*,hsa-miR-302d* hsa-miR-30a*, hsa-miR-30d*, hsa-miR-30e* hsa-miR-30a,hsa-miR-30c, hsa-miR-30d, hsa-miR-30b, hsa-miR-30e hsa-miR-330-5p,hsa-miR-326 hsa-miR-34a, hsa-miR-34c-5p, hsa-miR-449a, hsa-miR-449bhsa-miR-362-3p, hsa-miR-329 hsa-miR-374a, hsa-miR-374b hsa-miR-376a,hsa-miR-376b hsa-miR-378, hsa-miR-422a hsa-miR-379*, hsa-miR-411*hsa-miR-381, hsa-miR-300 hsa-miR-509-5p, hsa-miR-509-3-5phsa-miR-515-5p, hsa-miR-519e* hsa-miR-516b*, hsa-miR-516a-3phsa-miR-517a, hsa-miR-517c hsa-miR-518a-5p, hsa-miR-527 hsa-miR-518f,hsa-miR-518b, hsa-miR-518c, hsa-miR-518a-3p, hsa-miR-518d-3phsa-miR-519c-3p, hsa-miR-519b-3p, hsa-miR-519a hsa-miR-519c-5p,hsa-miR-519b-5p, hsa-miR-523*, hsa-miR-518f*, hsa-miR-526a,hsa-miR-520c-5p, hsa-miR-518e*, hsa-miR-518d-5p, hsa-miR-522*,hsa-miR-519a* hsa-miR-519e, hsa-miR-33b* hsa-miR-520a-5p, hsa-miR-525-5phsa-miR-520g, hsa-miR-520h hsa-miR-524-5p, hsa-miR-520d-5phsa-miR-525-3p, hsa-miR-524-3p hsa-miR-548b-5p, hsa-miR-548a-5p,hsa-miR-548c-5p, hsa-miR-548d-5p hsa-miR-7-1*, hsa-miR-7-2* hsa-miR-99a,hsa-miR-100, hsa-miR-99b

We have constructed an 8-mer LNA-antimiR against miR-21, miR-155 andmiR-122 (designated here as micromiR) that is fully LNA modified andphosphorothiolated (see FIG. 1 and Table 6). Our results from repeatedexperiments in MCF-7, HeLa, Raw and Huh-7 cells using a luciferasesensor plasmid for miR-21, miR-155 and miR-122 demonstrate that thefully LNA-modified short LNA-antimiRs are highly potent in antagonizingmicroRNAs.

TABLE 4 LNA_antimiR & MicromiR sequences and predicted T_(m)s SEQ ID #microRNA sequence T_(m) (° C.) 3204 miR-21 TcAGtCTGaTaAgCT 73 3205GATAAGCT 33 3206 miR-155 TcAcAATtaGCAtTA 63 3207 TAGCATTA 45    4miR-122 CcAttGTcaCaCtCC 73 3208 CACACTCC 62 Capital letters are LNAunits, such as beta-D-oxy LNA. Lower case letters are DNA units.Internucleoside linkages are preferably phosphorothioate. LNA cytosinesare all preferably methylated/5-methyl cytosine.

The melting temperatures can be assessed towards the mature microRNAsequence, using a synthetic microRNA oligonucleotide (typicallyconsisting of RNA nucleotides with a phosphodiester backbone). Typicallymeasured T_(m)s are higher than predicted T_(m)s when using LNAoligomers against the RNA target.

Example 4: Assessment of miR-21 Antagonism by SEQ ID #3205 LNA-antimiRin MCF-7 Cells Using a Luciferase Sensor Assay

In order to assess the efficiency of a fully LNA-modified 8-merLNA-antimiR (SEQ ID #3205) oligonucleotide in targeting and antagonizingmiR-21, luciferase sensor constructs were made containing a perfectmatch target site for the mature miR-21 and as control, a target sitewith two mutations in the seed (FIG. 6). In order to monitor microRNA-21inhibition, the breast carcinoma cell line MCF-7 was transfected withthe different luciferase constructs together with the miR-21 antagonistSEQ ID #3205 at varying concentrations in comparison with a 15-merLNA-antimiR SEQ ID #3204 against miR-21. After 24 hours, luciferaseactivity was measured.

Results:

As seen in FIG. 2, the new fully LNA-modified 8-mer LNA-antimiR (SEQ ID#3205) shows two-fold higher potency compared to SEQ ID #3204, as shownby de-repression of the luciferase activity. By contrast, the controlmiR-21 sensor construct with two mismatches in the miR-21 seed did notshow any de-repression of the firefly luciferase activity, therebydemonstrating the specificity of the perfect match miR-21 sensor inmonitoring miR-21 activity in cells. The de-repression of luciferaseactivity by the 8-mer LNA-antimiR is clearly dose-dependent, which isnot seen with SEQ ID #3204. Moreover, the new 8-mer is also much morepotent at lower doses than SEQ ID #3204.

To conclude, the 8-mer LNA-antimiR (SEQ ID #3205) shows significantlyimproved potency in inhibition of miR-21 in vitro compared to the 15-merLNA-antimiR SEQ ID #3204 targeting miR-21.

Materials and Methods:

Cell Line:

The breast carcinoma cell line MCF-7 was purchased from ATCC (#HTB-22M).MCF-7 cells were cultured in EMEM medium, supplemented with 10% fetalbovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

400.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, MCF-7 cells were transfected with 0.8 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector(SDS Promega) together with 1 μl Lipofectamine2000 (Invitrogen)according to manufacturer's instructions. After 24 hours, cells wereharvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, afterwhich cells were centrifugated for 5 min at 10.000 rpm. The supernatantwas discarded and 50 μl 1× Passive Lysis Buffer (Promega) was added tothe cell pellet, after which cells were put on ice for 30 min. The lysedcells were spinned at 10.000 rpm for 30 min after which 20 μl weretransferred to a 96 well plate and luciferase measurements wereperformed according to manufacturer's instructions (Promega).

Example 5: Assessment of miR-21 Antagonism by SEQ ID #3205 LNA-antimiRin HeLa Cells Using a Luciferase Sensor Assay

To further assess the efficiency of the fully LNA-modified 8-merLNA-antimiR SEQ ID #3205 in targeting miR-21, the cervix carcinoma cellline HeLa was also transfected with the previously described miR-21luciferase sensor constructs alongside SEQ ID #3205 at varyingconcentrations as described in the above section (FIG. 3).

Results:

The SEQ ID #3205 shows complete de-repression of the miR-21 luciferasesensor construct in HeLa cells already at 5 nM compared to SEQ ID #3204,which did not show complete de-repression until the highest dose (50nM). In addition, antagonism of miR-21 by the 8-mer SEQ ID #3205LNA-antimiR is dose-dependent. To demonstrate the specificity of themiR-21 luciferase sensor assay, a mismatched miR-21 target site (2mismatches in seed) was also transfected into HeLa cells, but did notshow any de-repression of the firefly luciferase activity.

To conclude, the fully LNA-modified SEQ ID #3205 shows significantlyimproved potency in inhibition of miR-21 in vitro, in both MCF-7 andHeLa cells compared to the 15-mer LNA-antimiR SEQ ID #3204.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24 well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 0.2 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.7 μl Lipofectamine2000 (Invitrogen) according tomanufacturer's instructions. After 24 hours, cells were harvested forluciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24 well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 6: Assessment of miR-155 Antagonism by SEQ ID #3207 LNA-antimiRin Mouse RAW Cells Using a Luciferase Sensor Assay

To ask whether a fully LNA-modified 8-mer LNA-antimiR can effectivelyantagonize miR-155, a perfect match target site for miR-155 was clonedinto the same luciferase vector (psiCHECK2) and transfected into themouse leukaemic monocyte macrophage RAW cell line. Because theendogenous levels of miR-155 are low in the RAW cell line, the cellswere treated with 100 ng/ml LPS for 24 hours in order to induce miR-155accumulation.

Results:

Luciferase measurements showed that the fully LNA-modified 8-merLNA-antimiR SEQ ID #3207 targeting miR-155 was similarly effective inantagonizing miR-155 compared to the 15-mer LNA-antimiR SEQ ID #3206(FIG. 4). Both LNA-antimirs showed a >50% de-repression of the miR-155luciferase sensor at 0.25 nM concentration and inhibited miR-155 in adose-dependent manner.

Conclusion:

These data further support the results from antagonizing miR-21, asshown in examples 1 and 2, demonstrating that a fully thiolated 8-merLNA-antimiR is highly potent in microRNA targeting.

Materials and Methods:

Cell Line:

The mouse leukaemic monocyte macrophage RAW 264.7 was purchased fromATCC (TIB-71). RAW cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500.000 cells were seeded per well in a 6 well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, MCF-7 cells were transfected with 0.3 ug miR-155 orempty psiCHECK2 vector together with 10 μl Lipofectamine2000(Invitrogen) according to manufacturer's instructions. In order toinduce miR-155 accumulation, LPS (100 ng/ml) was added to the RAW cellsafter the 4 hour incubation with the transfection complexes. Afteranother 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, afterwhich cells were centrifugated for 5 min at 2.500 rpm. The supernatantwere discarded and 50 μl 1× Passive Lysis Buffer (Promega) was added tothe cell pellet, after which cells were put on ice for 30 min. The lysedcells were spinned at 10.000 rpm for 30 min after which 20 μl weretransferred to a 96 well plate and luciferase measurements wereperformed according to manufacturer's instructions (Promega).

Example 7: Assessment of miR-122 Antagonism by SEQ ID #3208 LNA-antimiRin HuH-7 Cells Using a Luciferase Sensor Assay

The potency of the fully modified 8-mer LNA-antimiR SEQ ID #3208 againstmiR-122 was assessed in the human hepatoma cell line HuH-7. The HuH-7cells were transfected with luciferase sensor construct containing aperfect match miR-122 target site. After 24 hours luciferasemeasurements were performed (FIG. 5).

Results:

The fully LNA-modified 8-mer LNA-antimiR SEQ ID #3208 is more potentthan the 15-mer LNA-antimiR SEQ ID #4 at low concentration, as shown byde-repression of the miR-122 luciferase sensor. Both LNA-antimiRsinhibit miR-122 in a dose-dependent manner (FIG. 5).

Conclusion:

The fully LNA-modified 8-mer LNA-antimiR SEQ ID #3208 targeting miR-122shows improved potency in inhibition of miR-122 in vitro.

Materials and Methods:

Cell Line:

The human hepatoma cell line HuH-7 was a kind gift from R.Bartenschlager, Heidelberg. Huh-7 cells were cultured in EMEM medium,supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25ug/ml Gentamicin.

Transfection:

8.000 cells were seeded per well in a 96 well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HuH-7 cells were transfected with 57 ng miR-122 orempty psiCHECK2 vector together with 1 μl Lipofectamine2000(Invitrogen). After 24 hours, cells were harvested for luciferasemeasurements.

Lucierase Assay:

50 μl 1× Passive Lysis Buffer (Promega) was added to each well, afterwhich the 96 well plate was put on an orbital shaker for 30 min. To eachwell the Dual-luciferase Reporter assay system (Promega) was added andluciferase measurements were performed according to manufacturer'sinstructions (Promega).

Example 8. Assessment of miR-21 Antagonism by Comparing an 8-Mer (SEQ ID#3205) Versus a 15-Mer (SEQ ID #3204) LNA-antimiR in Human ProstateCarcinoma Cells (PC3)

We have previously shown (patent application 1051), that an 8-merLNA-antimiR that is fully LNA-modified and phosphorothiolated is able tocompletely de-repress the miR-21 luciferase reporter levels in the humancervix carcinoma cell line HeLa and partly de-repress the miR-21luciferase reporter levels in the human breast carcinoma cell lineMCF-7. We next extended this screening approach to the human prostatecancer cell line PC3. To assess the efficiency of the differentLNA-antimiR oligonucleotides against miR-21, luciferase reporterconstructs were generated in which a perfect match target site for themature miR-21 and a target site with two mismatches in the seed werecloned in the 3′UTR of Renilla luciferase gene (FIG. 7). In order tomonitor miR-21 inhibition, PC3 cells were transfected with the differentluciferase constructs together with the miR-21 antagonist SEQ ID #3205(8-mer) and for comparison with the 15-mer LNA-antimiR perfect match SEQID #3204 at varying concentrations. After 24 hours, luciferase activitywas measured.

Results:

The luciferase reporter experiments showed a dose-dependentde-repression of the luciferase miR-21 reporter activity with the 15-merLNA-antimiR against miR-21 (SEQ ID #3204). However, completede-repression of the luciferase reporter was not obtained even at thehighest concentrations (FIG. 7). In contrast, the cells that weretransfected with the 8-mer fully LNA substituted LNA-antimiR showedcomplete de-repression already at 1 nM, indicating significantlyimproved potency compared to the 15-mer LNA-antimiR. The luciferasecontrol reporter harboring a mismatch target site for miR-21 was notaffected by either LNA-antimiR, demonstrating high specificity of bothLNA-antimiRs.

Conclusion:

The micromer is far more potent than the 15-mer LNA-antimiR in targetingmiR-21 and has so far shown to be most potent in prostate carcinomacells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714). PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

100.000 cells were seeded per well in a 12-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with 0.3 μg miR-21 or emptypsiCHECK2 vector together with 1.2 μl Lipofectamine2000 (Invitrogen)according to manufacturer's instructions. Transfected was also varyingconcentrations of LNA-antimiRs. After 24 hours, cells were harvested forluciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 250 μl 1× Passive Lysis Buffer(Promega) was added to the wells. The plates were placed on a shaker for30 min., after which the cell lysates were transferred to eppendorftubes. The cell lysate was centrifugated for 10 min at 2.500 rpm afterwhich 20 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 9. Specificity Assessment of miR-21 Antagonism by an 8-MerLNA-antimiR

To investigate the specificity of our short LNA-antimiR targetingmiR-21, we designed an 8-mer mismatch control LNA-antimiR (SEQ ID #3218)containing 2 mismatches in the seed recognition sequence (see FIG. 8).The luciferase reporter constructs described in example 1 weretransfected into the human cervix carcinoma cell line HeLa together withthe LNA mismatch control oligo SEQ ID #3218 and its efficacy wascompared with the 8-mer LNA-antimiR (SEQ ID #3205) targeting miR-21.After 24 hours, luciferase activity was measured.

Results:

As shown in FIG. 8, transfection of the fully LNA-modified 8-merLNA-antimiR in HeLa cells resulted in complete de-repression of theluciferase miR-21 reporter already at 5 nM. In contrast, when the cellswere transfected with the 8-mer LNA mismatch control oligo, combinedwith the results obtained with the control miR-21 luciferase reporterhaving two mismatches in the miR-21 seed, these data demonstrate highspecificity of the fully LNA-substituted 8-mer LNA-antimiR in targetingmiR-21 in Hela cells.

Analysis of the miRBase microRNA sequence database showed that themiR-21 recognition sequence, of the LNA-antimiR SEQ ID #3205 is uniquefor microRNA-21. However, when decreasing the micromer length to 7 nt,it is not specific for only miR-21, since ath-miR-844, mmu-miR-590-3pand has-miR-590-3p are also targeted.

Conclusion:

Exchanging two nucleotide positions within the 8-mer LNA-antimiR withtwo mismatching nucleotides completely abolished the antagonizingactivity of the LNA-antimiR for miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, Ix NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 0.2 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.7 μl Lipofectamine2000 (Invitrogen) according tomanufacturer's instructions. Transfected was also varying concentrationsof LNA-antimiRs. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates wereput on an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96-well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 10. Assessment of the Shortest Possible Length of a FullyLNA-Modified LNA-antimiR that Mediates Effective Antagonism of miR-21

To further investigate the LNA-antimiR length requirements, we designeda 7-mer and a 6-mer LNA-antimiR targeting miR-21, both fullyLNA-modified and phosphorothiolated oligonucleotides. The miR-21luciferase reporter constructs were transfected into HeLa cells alongwith the LNA-antimiRs at varying concentrations. Luciferase measurementswere performed after 24 hours.

Results:

As seen in FIG. 9, the 7-mer LNA-antimiR mediates de-repression of themiR-21 luciferase reporter plasmid, but at lower potency compared to the8-mer LNA-antimiR (SEQ ID #3205). Nevertheless, a dose-dependent trendcan still be observed. By contrast, the 6-mer LNA-antimiR did not showany inhibitory activity.

Conclusion:

To conclude, the shortest possible length of an LNA-antimiR which isable to mediate miR-21 inhibition is 7 nucleotides. However, the 7-merLNA-antimiR is less potent compared to the 8-mer LNA-antimiR for miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24 well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 0.2 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.7 μl Lipofectamine2000 (Invitrogen) according tomanufacturer's instructions. Transfected was also varying concentrationsof LNA-antimiRs. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96-well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 11. Length Assessment of Fully LNA-Substituted LNA-antimiRsAntagonizing miR-21

Next, we investigated the effect of increasing the length from a 9-merto a 14-mer fully LNA substituted LNA-antimiRs on antagonizing miR-21 inHeLa cells. The resulting LNA-antimiRs were transfected into HeLa cellstogether with the miR-21 luciferase reporter constructs (FIG. 10).Luciferase measurements were performed after 24 hours.

Results:

The 9-mer LNA-antimiR SEQ ID #3211 (9-mer) showed dose-dependentde-repression of the miR-21 luciferase reporter which did not reachcomplete de-repression, as demonstrated for the 7-mer LNA-antimiR (SEQID #3210). Increasing the length to 10-mer to 14-mer (SEQ ID #3212, SEQID #3213 and SEQ ID #3214) decreased the potency as shown by lessefficient de-repression of the miR-21 reporter.

Conclusion:

As shown in FIG. 10, the longest fully LNA-modified andphosphorothiolated LNA-antimiR which is still able to mediate miR-21inhibition is a 9-mer LNA-antimiR SEQ ID #3211. However, it is clearlyless efficient than the 7-mer and 8-mer LNA-antimiRs.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 0.2 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 controlvector without target site together with 0.7 μl Lipofectamine2000(Invitrogen) according to manufacturer's instructions. Transfected wasalso varying concentrations of LNA-antimiRs. After 24 hours, cells wereharvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates wereput on an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96-well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 12. Determination of the Most Optimal Position for an 8-MerLNA-antimiR within the miR Target Recognition Sequence

Our experiments have shown that the most potent fully LNA-modifiedphosphorothiolated LNA-antimiR is 8 nucleotides in length. To assess themost optimal position for an 8-mer LNA-antimiR within the miR targetrecognition sequence, we designed four different fully LNA-modified8-mer LNA-antimiRs tiled across the mature miR-21 sequence as shown inFIG. 11. The different LNA-antimiRs were co-transfected together withthe miR-21 luciferase reporter constructs into HeLa cells. Luciferasemeasurements were performed after 24 hours.

Results:

The only LNA-antimiR that mediated efficient silencing of miR-21 asmeasured by the luciferase reporter was SEQ ID #3205, which targets theseed region of miR-21. Neither SEQ ID #3215 which was designed to coverthe 3′end of the seed (50% seed targeting) did not show any effect, nordid the other two LNA-antimiRs SEQ ID #3216 or SEQ ID #3217, which werepositioned to target the central region and the 3′end of the maturemiR-21, respectively.

Conclusion:

The only 8-mer LNA-antimiR mediating potent silencing of miR-21 is theone targeting the seed of the miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 0.2 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.7 μl Lipofectamine2000 (Invitrogen) according to themanufacturer's instructions. Transfected was also varying concentrationsof LNA-antimiRs. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 13. Validation of Interaction of the miR-21 Target Site in thePdcd4-3′-UTR and miR-21 Using the 8-Mer SEQ ID #3205 LNA-antimiR

The tumour suppressor protein Pdcd4 inhibits TPA-induced neoplastictransformation, tumour promotion and progression. Pdcd4 has also beenshown to be upregulated in apoptosis in response to different inducers.Furthermore, downregulation of Pdcd4 in lung and colorectal cancer hasalso been associated with a poor patient prognosis. Recently, Asanganiet al and Frankel et al showed that the Pdcd4-3′-UTR contains aconserved target site for miR-21, and transfecting cells with anantimiR-21, resulted in an increase in Pdcd4 protein. We thereforeconstructed a luciferase reporter plasmid, harboring 313 nt of the 3′UTRregion of Pdcd4 encompassing the aforementioned miR-21 target site,which was co-transfected together with different LNA-antimiRs into HeLacells. The different LNA-antimiRs were; SEQ ID #3205 (8-mer, perfectmatch) or SEQ ID #3218 (8-mer, mismatch). Luciferase measurements wereperformed after 24 hours.

Results:

As shown in FIG. 12, in cells transfected with the Pdcd4 3′UTRluciferase reporter and SEQ ID #3205, an increase in luciferase activitywas observed, indicating interaction between the Pdcd4 3′UTR and miR-21.However, transfecting the cells with the mismatch compound, SEQ ID#3218, no change in luciferase activity was observed, which was expectedsince the compound does not antagonize miR-21. When comparing the 8-merLNA-antimiR against two longer designed LNA-antimiRs, the short fullyLNA-modified and phosphorothiolated LNA-antimiR was significantly morepotent, confirming previous luciferase assay data.

Conclusion:

These data conclude that SEQ ID #3205, which antagonizes miR-21, canregulate the interaction between Pdcd4 3′UTR and miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 0.2 ugPdcd4-3′UTR/psiCHECK2 or empty psiCHECK2 vector together with 0.7 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. Varying concentrations of the LNA-antimiR oligonucleotideswere also transfected. After 24 hours, cells were harvested forluciferase measurements.

Lucierase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 14. Comparison of an 8-Mer LNA-antimiR (SEQ ID #3207) with a15-Mer LNA-antimiR (SEQ ID #3206) in Antagonizing miR-155 in Mouse RAWCells

To ask whether our approach of using short LNA-antimiRs could be adaptedto targeting other miRNAs we designed a fully LNA-modified 8-merLNA-antimiR against microRNA-155. A perfect match target site formiR-155 was cloned into the 3′UTR of the luciferase gene in the reporterplasmid psiCHECK2 and transfected into the mouse RAW macrophage cellline together with an 8-mer or a 15-mer LNA-antimiR. Because theendogenous levels of miR-155 are low in the RAW cell line, the cellswere treated with 100 ng/ml LPS for 24 hours in order to induce miR-155accumulation. After 24 hours, luciferase analysis was performed.

Results:

Luciferase measurements showed that the fully LNA-modified 8-merLNA-antimiR SEQ ID #3207 targeting miR-155 was similarly effective inantagonizing miR-155 compared to the 15-mer LNA-antimiR SEQ ID #3206(FIG. 13). Both LNA-antimiRs showed a >50% de-repression of the miR-155luciferase sensor at 0.25 nM concentration and inhibited miR-155 in adose-dependent manner.

Analysis of the miRBase microRNA sequence database showed that themiR-155 recognition sequence, of the LNA-antimiR SEQ ID #3207 is uniquefor microRNA-155. However, when decreasing the LNA-antimiR length to 7nt, it is not specific for only miR-155, mdv1-miR-M4 and kshv-miR-K12-11is also targeted.

Conclusion:

A fully LNA-modified and phosphorothiolated 8-mer LNA-antimiR is equallypotent compared with a 15-mer LNA-antimiR of a mixed LNA/DNA design inantagonizing miR-155. Thus, our approach of using short LNA-antimiRs canbe readily adapted to targeting of other miRNAs

Materials and Methods:

Cell Line:

The mouse macrophage RAW 264.7 cell line was purchased from ATCC(TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10%fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, RAW 264.7 cells were transfected with 0.3 ug miR-155perfect match/psiCHECK2 or empty psiCHECK2 vector together with 10 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. Transfected was also varying concentrations ofLNA-antimiRs. In order to induce miR-155 accumulation, LPS (100 ng/ml)was added to the RAW cells after the 4 hour incubation with thetransfection complexes. After another 24 hours, cells were harvested forluciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, afterwhich cells were spinned for 5 min at 2.500 rpm. The supernatant wasdiscarded and 50 μl 1× Passive Lysis Buffer (Promega) was added to thecell pellet, after which cells were put on ice for 30 min. The lysedcells were spinned at 10.000 rpm for 30 min after which 20 μl weretransferred to a 96-well plate and luciferase measurements wereperformed according to the manufacturer's instructions (Promega).

Example 15. Assessment of c/EBPβ Protein Levels as a Functional Readoutfor miR-155 Antagonism by Short LNA-antimiR (SEQ ID #3207)

As a functional readout for miR-155 antagonism by short LNA-antimiR (SEQID #3207) we determined the protein levels of a novel miR-155 target,c/EBPβ. The mouse macrophage RAW cell line was transfected together witheither an 8-mer (SEQ ID #3207) or a 15-mer (SEQ ID #3206) LNA-antimiR inthe absence or presence of pre-miR-155. As mismatch controls for the15-mer, SEQ ID #4 was used, which targets miR-122 and for the 8-mer SEQID #3205 was used, which targets miR-21. These two control miRNAs do notregulate c/EBPβ expression levels. LPS was used to induce miR-155accumulation and cells were harvested after 16 hours with LPS. c/EBPβhas three isoforms; LIP, LAP and LAP* that were detected by Western blotanalysis and the same membranes were re-probed with beta-tubulin asloading control.

Results:

Ratios were calculated for c/EBPβ LIP and beta-tubulin as indicated inFIG. 14. RAW cells that were transfected with the 15-mer LNA-antimiR andno pre-miR-155 all showed equal c/EBPβ LIP/beta-tubulin ratios, due toinhibition of miR-155 increases the c/EBPβ LIP levels (FIG. 14, leftpanel). By comparison, transfection of pre-miR-155 in RAW cells resultedin decreased c/EBPβ LIP levels as expected, if c/EBPβ was a miR-155target, as shown in lanes with protein extracts from RAW cells treatedwith no LNA or a mismatch. However, protein extracts from RAW cellstransfected with LNA-antimiR against miR-155, showed an increase ofc/EBPβ LIP levels. The same experiments were also carried out with the8-mer LNA-antimiR-155 (SEQ ID #3207) and as shown in FIG. 14 (rightpanel) comparable results to those with the 15-mer LNA-antimiR SEQ ID#3206 were obtained.

Conclusion:

Antagonism of miR-155 using either an 8-mer or a 15-mer LNA-antimiRleads to de-repression of the direct target c/EBPβ.

Materials and Methods:

Cell Line:

The mouse macrophage RAW 264.7 cell line was purchased from ATCC(TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10%fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to achieve 50% confluency the next day. On the dayof transfection, RAW 264.7 cells were transfected with 5 nmolpre-miR-155 (Ambion) and/or 5 nM LNA-antimiR together with 10 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. Transfected was also varying concentrations ofLNA-antimiRs. In order to induce miR-155 accumulation, LPS (100 ng/ml)was added to the RAW cells after the 4 hour incubation with thetransfection complexes. After 16 hours, cells were harvested for proteinextraction and western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubesand 250 μl lysis buffer (1×RIPA) was added. The cell lysate was placedon ice for 20 min and spinned at 10.000 rpm for 10 minutes. The proteinconcentration was measured with Coomassie Plus according to themanufacturer's instructions and 80 ug was loaded onto a 4-12% BIS-TRISgel. The membrane was incubated overnight at 4° C. with the primarymonoclonal mouse antibody C/EBP β (Santa Cruz) with a 1:100concentration. Immunoreactive bands were visualized with ECL Plus(Amersham).

Example 16. Antagonism of miR-106b by a Fully LNA-Modified 8-Mer (SEQ ID#3221) LNA-AntimiR

To confirm that our approach of using short LNA-antimiRs could beadapted to targeting of other miRNAs we designed a fully LNA-modified8-mer LNA-antimiR against microRNA-106b. A perfect match target site formiR-106b was cloned into the 3′UTR of the luciferase gene in the vector(psiCHECK2) and transfected into the human cervix carcinoma HeLa cellline together with a short LNA-antimiR (SEQ ID #3221) or with a 15-merLNA-antimiR (SEQ ID #3228) at varying concentrations. Luciferasemeasurements were performed after 24 hours.

Results:

Transfection of the 8-mer LNA-antimiR SEQ ID #3221 against miR-106bresulted in dose-dependent inhibition of miR-106b as shown byde-repression of the luciferase reporter, which was completelyde-repressed at 1 nM LNA-antimiR concentration (FIG. 15). Comparableresults were obtained using the 15-mer LNA-antimiR SEQ ID #3228demonstrating that an 8-mer LNA-antimiR is similarly potent to a 15-mer.

Conclusion:

Targeting of miR-106b in HeLa cells shows that an 8-mer fullyLNA-modified and phosphorotiolated LNA-antimiR is equally potentcompared with a 15-mer LNA/DNA mixmer LNA-antimiR.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

5.200 cells were seeded per well in a 96-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 57 ng miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.14 μl Lipofectamine2000 (Invitrogen) according to themanufacturer's instructions. Transfected was also varying concentrationsof LNA-antimiRs. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 30 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to eppendorf tubes and spinned at 10.000 rpm for 30 minafter which luciferase measurements were performed according to themanufacturer's instructions (Promega).

Example 17. Antagonism of miR-19a by a Fully LNA-Modified 8-Mer (SEQ ID#3222) LNA-antimir

To further confirm that our approach of using short LNA-antimiRs can bereadily adapted to targeting of other miRNAs we designed a fullyLNA-modified 8-mer LNA-antimiR against microRNA-19a. A perfect matchtarget site for miR-19a was cloned in the 3′UTR of the luciferase genein the psiCHECK2 vector. The reporter plasmid was transfected into thehuman cervix carcinoma HeLa cell line together with a short LNA-antimiR(SEQ ID #3222) or with a 15-mer LNA-antimiR (SEQ ID #3229) targetingmiR-19a at varying concentrations. Luciferase measurements wereperformed after 24 hours.

Results:

As shown in FIG. 16, transfection of the 15-mer LNA-antimiR SEQ ID #3229into HeLa efficiently antagonizes miR-19a as demonstrated by completede-repression at 1 nM LNA-antimiR concentration. By comparison,transfection of the 8-mer LNA-antimiR SEQ ID #3222 resulted in effectivemiR-19a antagonism already at 0.5 nM concentration, indicating that this8-mer LNA-antimiR is at least equally potent compared with a 15-merLNA-antimiR in HeLa cells.

Conclusion:

Targeting of miR-19a in HeLa cells shows that an 8-mer fullyLNA-modified and phosphorothiolated LNA-antimiR is at least equallypotent compared with a 15-mer LNA/DNA mixmer LNA-antimiR.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

5.200 cells were seeded per well in a 96-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 57 ng miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.14 μl Lipofectamine2000 (Invitrogen) according tomanufacturer's instructions. Transfected was also varying concentrationsof LNA-antimiRs. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 30 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to eppendorf tubes and spinned at 10.000 rpm for 30 minafter which luciferase measurements were performed according to themanufacturer's instructions (Promega).

Example 18. Targeting of a microRNA Family Using Short, FullyLNA-Substituted LNA-antimiR

Next, we investigated whether it is possible to target a microRNA familyusing a single short 7-mer LNA-antimiR complementary to the seedsequence that is common for all family members (see FIG. 17). In thisexperiment, we focused on miR-221 and miR-222 that are overexpressed insolid tumors of the colon, pancreas, prostate and stomach. It has alsobeen shown that miR-221 and miR-222 are the most significantlyupregulated microRNAs in glioblastoma multiforme. Furthermore,overexpression of miR-221 and miR-222 may contribute to the growth andprogression of prostate carcinoma, at least in part by blocking thetumor suppressor protein p27. A perfect match target site for bothmiR-221 and miR-222, respectively, was cloned into the 3′UTR of theluciferase gene resulting in two reporter constructs. These constructswere then transfected either separate or combined into the prostatecarcinoma cell line, PC3. In addition to the 7-mer, targeting bothmiR-221 and miR-222, we also co-transfected a 15-mer LNA-antimiR (15mer)targeting either miR-221 (SEQ ID #3223) or miR-222 (SEQ ID #3224), eachtransfected separately or together (see FIG. 18 left).

Results:

As shown in FIG. 18, transfection of PC3 cells with the LNA-antimiR SEQID #3223 against miR-221 resulted in efficient inhibition of miR-221 at1 nM LNA-antimiR concentration. An inhibitory effect is also observedwhen using the luciferase reporter plasmid for miR-222 as well as whenco-transfecting both luciferase reporters for miR-221 and miR-222simultaneously into PC3 cells. This inhibitory effect is most likely dueto the shared seed sequence between miR-221 and miR-222. Similarly,transfection of PC3 cells with the LNA-antimiR SEQ ID #3224 againstmiR-222 resulted in efficient inhibition of miR-222 at 1 nM LNA-antimiRconcentration as shown by complete de-repression of the luciferasereporter for miR-222. An inhibitory effect is also observed when usingthe luciferase reporter plasmid for miR-222 as well as whenco-transfecting both luciferase reporters for miR-221 and miR-222simultaneously into PC3 cells. Co-tranfection of both LNA-antimiRcompounds SEQ ID #3223 and SEQ ID #3224 against miR-221 and miR-222,respectively, (see FIG. 18 left), resulted in effective inhibition ofboth miRNAs as shown by complete de-repression of the luciferasereporter plasmids both when separately transfected and whenco-transfected into PC3 cells. Interestingly, transfection of a singlefully LNA-modified 7-mer LNA-antimiR (SEQ ID #3225) targeting the seedsequence of miR-221 and miR-222 into PC3 cells resulted in efficient,dose-dependent antagonism of miR-221 and miR-222 simultaneously as shownby complete de-repression of the luciferase reporter plasmids both whenseparately transfected and when co-transfected into PC3 cells. Thisdemonstrates that a single, short LNA-substituted LNA-antimiR caneffectively target seed sequences thereby antagonizing entire microRNAfamilies simultaneously. Analysis of the miRBase microRNA sequencedatabase showed that the miR-221/222 seed recognition sequence, of theLNA-antimiR SEQ ID #3225 is unique for both miRNAs.

Conclusion:

Our results demonstrate that LNA enables design and synthesis of shortfully LNA-substituted LNA-antimiR oligonucleotides that can effectivelytarget microRNA seed sequences thereby antagonizing entire microRNAfamilies simultaneously.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714) PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

100.000 cells were seeded per well in a 12-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with 0.3 ug of luciferasereporter plasmid for miR-221 or for miR-222 or with empty psiCHECK2vector without miRNA target site as control together with 1.2 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 250 μl× Passive Lysis Buffer(Promega) was added to the wells. The plates were placed on a shaker for30 min., after which the cell lysates was transferred to eppendorftubes. The cell lysate was spinned for 10 min at 2.500 rpm after which20 μl were transferred to a 96-well plate and luciferase measurementswere performed according to the manufacturer's instructions (Promega).

Example 19. Assessment of p27 Protein Levels as a Functional Readout forAntagonism of the miR-2211222 family by the 7-mer SEQ ID #3225LNA-antimiR

Previous work has shown (le Sage et al. 2007, Galardi et al. 2007) thatmiR-221 and miR-222 post-transcriptionally regulate the expression ofthe tumour suppressor gene p27, which is involved in cell cycleregulation. In these studies, down-regulation of miR-221 and miR-222 wasshown to increase expression levels of p27. Thus, as a functionalreadout for antagonism of the miR-221/222 family by the 7-mer SEQ ID#3225 LNA-antimiR we determined the protein levels of p27 aftertransfection of the LNA-antimiR SEQ ID #3225 into PC3 cells incomparison with an 8-mer LNA mismatch control. After 24 hours the cellswere harvested for western blot analysis (FIG. 19).

Results:

As shown in FIG. 19, transfection of the 7-mer LNA-antimiR SEQ ID #3225targeting the seed sequence in miR-221 and miR-222 resulted indose-dependent increase of the p27 protein levels compared to eitheruntransfected or LNA mismatch control transfected PC3 cells. Theseresults clearly demonstrate that the 7-mer LNA-antimiR is able toeffectively antagonize the miR-221/222 family leading to de-repressionof the direct target p27 at the protein level.

Conclusion:

A fully LNA-modified 7-mer LNA-antimiR targeting the seed sequence inthe miR-221/222 family effectively antagonized both miRNAs leading tode-repression of the direct target p27 at the protein level.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714) PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with LNA-antimiRs at varyingconcentrations with Lipofectamine2000. Cells were harvested after 24hours for protein extraction and western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubesand 250 μl lysis buffer (1×RIPA) was added. The cell lysate was placedon ice for 20 min, then spinned at 10.000 rpm for 10 minutes. Theprotein concentration was measured with Coomassie Plus according to themanufacturer's instructions and 100 ug was loaded onto a 4-12% BIS-TRISgel. The membrane was incubated overnight at 4° C. with the primarymonoclonal mouse antibody p27 (BD Biosciences) at a 1:1000 dilution.Immunoreactive bands were visualized with ECL Plus (Amersham).

Example 20. Duplex Melting Temperatures (T_(m)) of the LNA-antimiRs

As shown in Table 5, T_(m) values increase with increasing the length ofshort fully modified LNA-antimiRs (see T_(m) values for SEQ ID #3205,SEQ ID #3209-3214 in Table 7). Most optimal inhibitory effect wasachieved with the 8-mer LNA-antimiR SEQ ID #3205 against miR-21, whereasthe very low T_(m) of the 6-mer SEQ ID #3209 is most likely notsufficient to mediate antagonism of the miR-21 target. On the otherhand, increasing the length beyond a 10-mer (SEQ ID #3212) significantlyincreases the T_(m), while simultaneously decreasing the inhibitoryactivity as measured using the luciferase miR-21 reporter, which is mostlikely due to high propensity of the fully modified 12- and 14-merLNA-antimiRs to form homodimers. The experiments using a sliding windowof fully LNA-modified 8-mer LNA-antimirs across the mir-21 recognitionsequence clearly demonstrate that in addition to adequate T_(m) value ofthe LNA-antimiR, the seed region is most critical for miRNA functionand, thus, the most optimal region to be targeted by an LNA-antimiR.

TABLE 5 T_(m) values for miR-21 LNA-antimiRs, measured against acomplementary RNA oligonucleotide SEQ ID micro- Length Measured T_(m) #RNA (bp) Sequence (RNA) ° C. 3205 miR-21  8 5′-GATAAGCT-3′ 64.0 3209miR-21  6 5′-TAAGCT-3′ 32.0 3210 miR-21  7 5′-ATAAGCT-3′ 45.0 3211miR-21  9 5′-TGATAAGCT-3′ 65.0 3212 miR-21 10 5′-CTGATAAGCT-3′ 63.0 3213miR-21 12 5′-GTCTGATAAGCT-3′ 86.8 3214 miR-21 14 5′-CAGTCTGATAAGCT-3′89.9 3215 miR-21  8 5′-TCTGATAA-3′ 56.0 3216 miR-21  8 5′-ATCAGTCT-372.0 3217 miR-21  8 5′-TCAACATC-3 48.0Conclusion:

The T_(m) values along with experimental data obtained with luciferasereporters show that potent antagonism by LNA-antimiR is not onlydependent on T_(m) but also depends on the positioning of theLNA-antimiR within the microRNA recognition sequence.

Materials and Methods:

T_(m) Measurements:

The oligonucleotide:miR-21 RNA duplexes were diluted to 3 μM in 500 μlRNase free H₂O and mixed with 500 μl 2× T_(m)-buffer (200 mM NaCl, 0.2mM EDTA, 20 mM Na-phosphate, pH 7.0). The solution was heated to 95° C.for 3 min and then allowed to anneal in RT for 30 min. The duplexmelting temperatures (T_(m)) were measured on a Lambda 40 UV/VISSpectrophotometer equipped with a Peltier temperature programmer PTP6using PE Templab software (Perkin Elmer). The temperature was ramped upfrom 20° C. to 95° C. and then down to 25° C., recording absorption at260 nm. First derivative and the local maximums of both the melting andannealing were used to assess the duplex melting temperatures.

Example 21. Assessment of miR-21 Antagonism by Comparing an 8-Mer (SEQID #3205) Versus a 15-Mer (SEQ ID #3204) LNA-antimiR in HumanHepatocytic Cell Line HepG2

We have previously shown in this application, that an 8-mer LNA-antimiRthat is fully LNA-modified and phosphorothiolated effectivelyantagonizes miR-21 in the human cervix carcinoma cell line HeLa, thehuman breast carcinoma cell line MCF-7 and the human prostate cancercell line PC3. We extended this screening approach to the humanhepatocellular cancer cell line HepG2. To assess the efficiency of the8-mer LNA-antimiR oligonucleotide against miR-21, luciferase reporterconstructs were generated in which a perfect match target site for themature miR-21 was cloned into the 3′UTR of the Renilla luciferase gene.In order to monitor miR-21 inhibition, HepG2 cells were transfected withthe luciferase constructs together with the miR-21 antagonist SEQ ID#3205 (8-mer) and for comparison of specificity with the 8-merLNA-antimiR mismatch (SEQ ID #3218) and for comparison of potencytogether with the 15-mer (SEQ ID #3204) at varying concentrations. After24 hours, luciferase activity was measured.

Results:

The luciferase reporter experiments showed a dose-dependentde-repression of the luciferase miR-21 reporter activity with the 15-merLNA-antimiR against miR-21 (SEQ ID #3204). However, completede-repression of the luciferase reporter was not obtained, not even atthe higher concentrations (FIG. 20). In contrast, the cells that weretransfected with the 8-mer fully LNA modified LNA-antimiR (SEQ ID #3205)showed complete de-repression already at 5 nM, indicating significantlyimproved potency compared to the 15-mer LNA-antimiR. Comparing thespecificity of the 8-mer perfect match and the 8-mer mismatch, themismatch LNA-antimiR (SEQ ID #3218) did not show any de-repression atall, demonstrating high specificity of the LNA-antimiR compound againstmiR-21.

Conclusion:

The 8-mer (SEQ ID #3205) is more potent than the 15-mer LNA-antimiR intargeting miR-21 and antagonism of miR-21 by SEQ ID #3205 is specific.

Materials and Methods:

Cell Line:

The human hepatocytic HepG2 cell line was purchased from ECACC(#85011430). HepG2 cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

650.000 cells were seeded per well in a 6-well plate and reversetransfection were performed. HepG2 cells were transfected with 0.6 μgmiR-21 or empty psiCHECK2 vector together with 2.55 μl Lipofectamine2000(Invitrogen) according to manufacturer's instructions. Transfected wasalso varying concentrations of LNA-antimiRs. After 24 hours, cells wereharvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 300 μl 1× Passive Lysis Buffer(Promega) was added to the wells. The plates were placed on a shaker for30 min., after which the cell lysates were transferred to eppendorftubes. The cell lysate was centrifugated for 10 min at 2.500 rpm afterwhich 50 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to the manufacturer's instructions(Promega).

Example 22. Validation of Interaction of the miR-21 Target Site in thePdcd4 3′UTR and miR-21 Using the 8-Mer SEQ ID #3205 LNA-antimiR in HumanHepatocellular Cell Line Huh-7

The tumour suppressor protein Pdcd4 inhibits tumour promotion andprogression. Furthermore, downregulation of Pdcd4 in lung and colorectalcancer has also been associated with poor patient prognosis. Recently,Asangani et al (Oncogene 2007) and Frankel et al (J Biol Chem 2008)showed that the Pdcd4 3′UTR contains a conserved target site for miR-21,and transfecting cells with an antimiR-21, resulted in an increase inPdcd4 protein. We therefore constructed a luciferase reporter plasmid,harboring 313 nt of the 3′UTR region of Pdcd4 encompassing theaforementioned miR-21 target site, which was co-transfected togetherwith different LNA-antimiRs and pre-miR-21 (10 nM) into Huh-7 cells. Thedifferent LNA-antimiRs were; SEQ ID #3205 (8-mer, perfect match), SEQ ID#3218 (8-mer, mismatch) and SEQ ID #3204 (15-mer, DNA/LNA mixmer).Luciferase measurements were performed after 24 hours.

Results:

As shown in FIG. 21, cells transfected with the Pdcd4 3′UTR luciferasereporter and SEQ ID #3205, an increase in luciferase activity wasobserved, indicating interaction between the Pdcd4 3′UTR and miR-21.However, transfecting the cells with the mismatch compound, SEQ ID#3218, no change in luciferase activity was observed, which was expectedsince the compound does not antagonize miR-21. When comparing the 8-merLNA-antimiR against the 15-mer LNA-antimiR (SEQ ID #3204), the shortfully LNA-modified and phosphorothiolated LNA-antimiR was significantlymore potent, confirming previous data.

Materials and Methods:

Cell Line:

The human hepatoma cell line Huh-7 was a kind gift from R.Bartinschlager (Dept Mol Virology, University of Heidelberg). Huh-7cells were cultured in DMEM medium, supplemented with 10% fetal bovineserum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

11.000 cells were seeded per well in a 96-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection, Huh-7 cells were transfected with 20 ng Pdcd43′UTR/psiCHECK2 or empty psiCHECK2 vector together with 10 nM pre-miR-21(Ambion) and 0.14 j Lipofectamine2000 (Invitrogen) according to themanufacturer's instructions. Varying concentrations of the LNA-antimiRoligonucleotides were also transfected. After 24 hours, cells wereharvested for luciferase measurements.

Luciferase Assay:

Cells were washed and 30 μl 1× Passive Lysis Buffer (Promega) was addedto each well, after which the 96-well plates was put on an orbitalshaker. After 30 min., 50 μl luciferase substrate dissolved inLuciferase Assay Buffer II (Dual-Luciferase Reporter Assay System fromPromega, Cat# E1910) was added to the wells with lysated cells andluciferase measurements were performed according to the manufacturer'sinstructions (Promega).

Example 23. Assessment of Pdcd4 Protein Levels as a Functional Readoutfor miR-21 Antagonism by the 8-Mer LNA-antimiR (SEQ ID #3205)

In addition, we also transfected HeLa cells with SEQ ID #3205 (perfectmatch), SEQ ID #3218 (mismatch), SEQ ID #3219 (scrambled) and analyzedPdcd4 protein levels after 24 hours with Western blot (FIG. 22). Asshown, in the protein extracts from cells where SEQ ID #3205 had beenadded, the Pdcd4 protein levels increase, due to antagonism of mir-21 bySEQ ID #3205 in contrast to the two control LNA oligonucleotides.

Conclusion:

Antagonism of miR-21 using an 8-mer (SEQ ID #3205) leads to derepressionof the direct target Pdcd4□ntagonism of miR-21

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

200.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 5 nM LNAoligonucleotides and 2.5 μg/ml Lipofectamine2000 (Invitrogen) accordingto the manufacturer's instructions. After 24 hours, cells were harvestedfor Western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubesand 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed onice for 20 min and spinned at 10.000 rpm for 10 minutes. Equal amounts(15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteinswere transferred to a nitrocellulose membrane using iBlot (Invitrogen)according to manufacturers instructions. The membrane was incubatedovernight at 4° C. with the primary affinity purified rabbit serumantibody Pdcd4 (Rockland) with a 1:2000 concentration. As control,anti-beta tubulin antibodies (Thermo Scientific) were used at a 1:5000dilution. Immunoreactive bands were visualized with ECL Plus (Amersham).

Example 24. Assessment of Potential Hepatotoxicity of the 8-Mer PerfectMatch LNA-antimiR SEQ ID #3205 and the LNA Mismatch Control SEQ ID #3218

Each compound was injected into female NMRI mice, at doses of 25 mg/kg,5 mg/kg and 1 mg/kg, every other day for 2 weeks. The animals weresacrificed and serum was collected from whole blood for ALT and ASTanalyses. As seen in FIG. 23, the ALT and AST levels were not elevatedfor SEQ ID #3205 compared to saline or SEQ ID #3218 (mismatch control).However, one mouse showed increased levels (marked red), since the serumsamples were contaminated with red blood cells, which contain 6-8 timeshigher levels of ALT and AST compared to plasma. The mice that received5 mg/kg and 1 mg/kg were also analyzed for ALT and AST levels and showedno changes compared to saline treated control animals (data not shown).

Materials and Methods:

Experimental Design:

Conc. Gr. Animal No. of Compound at dose vol. no. ID no. mice Dose levelper day 10 ml/kg Adm. Route Dosing 1  1-10 10 NaCl — i.v 0, 2, 4, 7, 90.9% 2 11-15 5 SEQ ID # 3205 2.5 mg/ml i.v 0, 2, 4, 7, 9 25 mg/kg 316-20 5 SEQ ID # 3205 0.5 mg/ml i.v 0, 2, 4, 7, 9 5 mg/kg 4 21-25 5 SEQID # 3205 0.1 mg/ml i.v 0, 2, 4, 7, 9 1 mg/kg 5 26-30 5 SEQ ID # 32302.5 mg/ml i.v 0, 2, 4, 7, 9 25 mg/kg 6 31-35 5 SEQ ID # 3230 0.5 mg/mli.v 0, 2, 4, 7, 9 5 mg/kgSacrifice:

The animals was sacrificed by cervical dislocation.

Sampling of Serum for ALT/AST:

The animals were anaesthetised with 70% CO₂-30% O₂ before collection ofretro orbital sinus blood. The blood was collected into S-monovetteSerum-Gel vials. The serum samples were harvested and stored from eachindividual mouse. The blood samples were stored at room temperature fortwo hours and thereafter centrifuged 10 min, 3000 rpm, at room temp. Theserum fractions were harvested into Eppendorf tubes on wet ice.

ALT and AST Measurements:

ALT and AST measurements were performed in 96-well plates using ALT andAST reagents from ABX Pentra (A11A01627—ALT, A11A01629—AST) according tothe manufacturer's instructions. In short, serum samples were diluted2.5 fold with H₂O and each sample was assayed in duplicate. Afteraddition of 50 μl diluted sample or standard (multical from ABXPentra—A11A01652) to each well, 200 μl of 37° C. AST or ALT reagent mixwas added to each well. Kinetic measurements were performed for 5 minwith an interval of 30 s at 340 nm and 37° C.

Example 25. Assessment of PU.1 Protein Levels as a Functional Readoutfor miR-155 Antagonism by Short LNA-antimiR (SEQ ID #3207)

We have previously shown that the 8-mer (SEQ ID #3207) antagonizingmiR-155 leads to derepression of the miR-155 target c/EBPbeta in themouse macrophage RAW cells. To further verify the potency of SEQ ID#3207 we determined the protein levels of another miR-155 target, PU.1As a functional readout for miR-155 antagonism by short LNA-antimiR (SEQID #3207) we performed Western blot. The antagonism was verified in thehuman monocytic THP-1 cell line which was transfected together witheither an 8-mer (SEQ ID #3207) perfect match or a 8-mer control LNA inthe absence or presence of pre-miR-155. LPS was used to induce miR-155accumulation and cells were harvested after 24 hours.

Results:

THP-1 cells that were transfected with pre-miR-155 shows a decrease inPU.1 levels (FIG. 24). Transfecting the cells with the fullyLNA-modified and phosphorothiolated SEQ ID #3207 effectively antagonizesmiR-155, leading to unaltered levels of PU.1 protein. By comparison,transfecting the cells with an 8-mer LNA control, PU.1 levels decreased,indicating that antagonism of miR-155 by SEQ ID #3207 LNA-antimiR isspecific.

Conclusion:

Antagonism of miR-155 using an 8-mer leads to de-repression of thedirect target PU.1 in human THP-1 cells.

Materials and Methods:

Cell Line:

The human monocytic THP-1 cell line was purchased from ECACC(#88081201). THP-1 cells were cultured in RPMI with L-glutamine,supplemented with 10% fetal bovine serum.

Transfection:

200.000 cells were seeded per well in a 12-well plate the day before. Onthe day of transfection, THP-1 cells were transfected with 5 nmolpre-miR-155 (Ambion) and/or 5 nM LNA-antimiR together withLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. LPS (100 ng/ml) was added to the cells after the 4 hourincubation with the transfection complexes. After 24 hours, cells wereharvested for protein extraction and western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubesand 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed onice for 20 min and spinned at 10.000 rpm for 10 minutes. Equal amounts(15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteinswere transferred to a nitrocellulose membrane using iBlot (Invitrogen)according to manufacturers instructions The membrane was incubatedovernight at 4° C. with the rabbit monoclonal PU.1 antibody (CellSignaling) with a 1:2000 concentration. As equal loading, Tubulin(Thermo Scientific) was used at a 1:5000 dilution. Immunoreactive bandswere visualized with ECL Plus (Amersham).

Example 26. Assessment of p27 Protein Levels as a Functional Readout forAntagonism of the miR-2211222 Family by the 7-Mer SEQ ID #3225LNA-antimiR

Previous work has shown (le Sage et al. 2007, Galardi et al. 2007) thatmiR-221 and miR-222 post-transcriptionally regulate the expression ofthe tumour suppressor gene p27, which is involved in cell cycleregulation. In these studies, down-regulation of miR-221 and miR-222 wasshown to increase expression levels of p27. Thus, as a functionalreadout for antagonism of the miR-221/222 family by the 7-mer SEQ ID#3225 LNA-antimiR we determined the protein levels of p27 aftertransfection of the LNA-antimiR SEQ ID #3225 into PC3 cells.

Results:

As shown in FIG. 25, transfection of the 7-mer LNA-antimiR SEQ ID #3225targeting the seed sequence of miR-221 and miR-222 resulted indose-dependent increase of the p27 protein levels compared to eitheruntransfected or our LNA scrambled control transfected PC3 cells. Theseresults clearly demonstrate that the 7-mer LNA-antimiR is able toeffectively antagonize the miR-221/222 family leading to de-repressionof the direct target p27 at the protein level at concentrations as lowas 5 nM.

Conclusion:

A fully LNA-modified 7-mer LNA-antimiR targeting the seed sequence inthe miR-221/222 family at 5 nM can effectively antagonize both miRNAsleading to de-repression of the direct target p27 at protein level.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714). PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with LNA-oligonucleotides atvarying concentrations (see FIG. 25) with Lipofectamine2000. Cells wereharvested after 24 hours for protein extraction and western blotanalysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubesand 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed onice for 20 min, then spinned at 10.000 rpm for 10 minutes. Equal amounts(15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteinswere transferred to a nitrocellulose membrane using iBlot (Invitrogen)according to manufacturers instructions. The membrane was incubatedovernight at 4° C. with the primary monoclonal mouse antibody p27 (BDBiosciences) at a 1:1000 dilution. As loading control, Tubulin (ThermoScientific) was used at a 1:5000 dilution. Immunoreactive bands werevisualized with ECL Plus (Amersham).

Example 27. Knock-Down of miR-2211222 by the 7-Mer SEQ ID #3225LNA-antimiR Reduces Colony Formation of PC3 Cells

A hallmark of cellular transformation is the ability for tumour cells togrow in an anchorage-independent way in semisolid medium. We havetherefore performed soft agar assay which is a phenotypic assay that isrelevant for cancer, given that it measures the decrease of tumourcells. We transfected SEQ ID #3225 (perfect match) and SEQ ID #3231(scrambled) into PC3 cells, and after 24 hours plated cells in softagar. Colonies were counted after 12 days. We show in FIG. 26 thatinhibition of miR-221 and miR-222 by SEQ ID #3225 can reduce the amountof colonies growing in soft agar compared to the scrambled controlLNA-antimiR, indicating decrease of tumour cells.

Conclusion:

The 7-mer (SEQ ID #3225) targeting the miR-221/222 family reduces thenumber of colonies in soft agar, indicating proliferation arrest of PC3cells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714). PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250,000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with 25 nM of different LNAoligonucleotides with Lipofectamine2000.

Clonogenic Growth in Soft Agar:

2.5×10³ PC3 cells were seeded in 0.35% agar on the top of a base layercontaining 0.5% agar. Cells were plated 24 hours after transfection.Plates were incubated in at 37° C., 5% CO₂ in a humified incubator for12 days and stained with 0.005% crystal violet for 1 h, after whichcells were counted. The assay was performed in triplicate.

Example 28: Assessment of Let-7 Antagonism by 6-9-Mer LNA-antimiRs inHuh-7 Cells Transfected with Let-7a Precursor miRNA, and a LuciferaseSensor Assay

In order to assess the efficiency of fully LNA-modified 6-9-meroligonucleotides in targeting and antagonizing the let-7 family ofmiRNAs, a luciferase sensor construct was made, containing some 800 bpof the HMGA2 3′UTR. The sequence cloned into the vector contains fourout of seven functional let-7 binding sites (sites 2-5), as previouslydemonstrated by Mayr et al. (Science, 2007) and Lee and Dutta (GenesDev, 2007). In order to monitor let-7 inhibition, the hepatocellularcarcinoma cell line Huh-7 (with low to non-existing levels of endogenouslet-7) was transfected with the luciferase sensor construct, with let-7aprecursor miRNA, and with the 6-9 mer let-7 antagonists SEQ ID #3232,-3233, -3227, -3234, -3235; see FIG. 27) at increasing concentrations.The 6-9-mer LNA-antimiRs were compared with SEQ ID #3226, a 15-meragainst let-7a as a positive control. After 24 hours, luciferaseactivity was measured.

Results:

As seen in FIG. 28, the fully LNA-modified 8- and 9-mer LNA-antimiRs(SEQ ID #3227, SEQ ID #3234, and SEQ ID #3235) show similar potencies inde-repressing the let-7 targets in the luciferase sensor assay, as thepositive control 15-mer SEQ ID #3226. Full target de-repression forthese highly potent compounds is achieved already at 1-5 nM, whereas the7-mer SEQ ID #3233 needs to be present at slightly higher concentrations(10 nM) to generate the same effect. However, the 6-mer SEQ ID #3232shows no effect even at as high concentrations as 50 nM. Thede-repression of luciferase activity by the 7-9- and the 15-merLNA-antimiRs is dose-dependent, which is particularly clear in the caseof the slightly less potent SEQ ID #3233.

Conclusion:

To conclude, the 8-9-mer LNA-antimiRs (SEQ ID #3227, SEQ ID #3234, andSEQ ID #3235) show equal antagonist potencies in inhibition of let-7a invitro compared to the 15-mer LNA-antimiR SEQ ID #3226 targeting let-7a.A potent effect, albeit at slightly higher concentrations is also seenfor the 7-mer SEQ ID #3233, whereas a 6-mer has no effect at testedconcentrations.

Materials and Methods:

Cell Line:

The hepatocellular carcinoma cell line Huh-7 was a kind gift from R.Bartinschlager (Dept Mol Virology, University of Heidelberg). Huh-7cells were cultured in DMEM medium, supplemented with 10% fetal bovineserum, 2 mM Glutamax, Ix NEAA and 25 ug/ml Gentamicin.

Transfection:

8,000 cells were seeded per well in a 96-well plate the day beforetransfection in order to receive 60-80% confluency the next day. On theday of transfection, Huh-7 cells in each well were transfected with 20ng HMGA2 3′UTR/psiCHECK2 plasmid, let-7a precursor miRNA (Dharmacon; 10nM end-concentration), LNA-antimiRs SEQ ID #3232, -3233, -3227, -3234,-3235, -3226; 0-50 nM end concentrations) together with 0.17 μlLipofectamine2000 (Invitrogen) according to manufacturer's instructions.After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

Growth media was discarded and 30 μl 1× Passive Lysis Buffer (Promega)was added to each well. After 15-30 minutes of incubation on an orbitalshaker, renilla and firefly luciferase measurements were performedaccording to manufacturer's instructions (Promega).

Example 29: Assessment of Entire Let-7 Family Antagonism by 8-, and15-Mer LNA-antimiRs in Huh-7 Cells Transfected with a Luciferase SensorAssay

In order to assess the efficiency of a fully LNA-modified 8-meroligonucleotide in antagonizing the entire let-7 family of miRNAs, thesame luciferase sensor construct as described in the previous examplewas used. Again, Huh-7 cells (with low to non-existing levels ofendogenous let-7) were transfected with the sensor construct, with oneof the family-representative let-7a, let-7d, let-7e, or let-7iprecursors, and with the antagonist SEQ ID #3227 at increasingconcentrations. The 8-mer LNA-antimiR was compared to SEQ ID #3226, a15-mer against let-7a as a positive and potent control. After 24 hours,luciferase activity was measured.

Results:

As seen in FIG. 29 the fully LNA-modified 8-mer LNA-antimiRs (SEQ ID#3227) show similar potencies in de-repressing the various let-7 targetsin the luciferase sensor assay, as the positive control 15-mer SEQ ID#3226. Nearly full target de-repression for the 8-mer is achievedalready at 0.5-1 nM, except in the case with let-7e premiR (FIG. 29C),to which only 7 out of 8 nucleotides of SEQ ID #3227 hybridizes to thetarget. However, despite the terminal mismatch in this case, SEQ ID#3227 generates full target de-repression at 5 nM. The positive control15-mer shows potent antagonism of all precursors and gives nearly fullde-repression at 0.5 nM. The de-repression of luciferase activity byboth the 8- and the 15-mer LNA-antimiRs is clearly dose-dependent, asseen in all four panels (FIG. 29A-D).

Conclusion:

To conclude, the 8-mer LNA-antimiR (SEQ ID #3227), is a potentantagonist against four representative let-7 family members in vitro,and thus likely against the entire family. Compared to a 15-mer positivecontrol antagonist, SEQ ID #3226, the 8-mer is equally potent for threeof four targets, and slightly less potent for the fourth target, let-7e,explained by a terminal mismatch in this case.

Materials and Methods:

Cell Line:

The hepatocellular carcinoma cell line Huh-7 was a kind gift from R.Bartinschlager (Dept Mol Virology, University of Heidelberg). Huh-7cells were cultured in DMEM medium, supplemented with 10% fetal bovineserum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfectin: 8,000 cells were seeded per well in a 96-well plate the daybefore transfection in order to receive 60-80% confluency the next day.On the day of transfection, Huh-7 cells in each well were transfectedwith 20 ng HMGA2 3′UTR/psiCHECK2 plasmid, with let-7a, -7d, -7e, or -7iprecursor miRNA (Dharmacon; 10 nM end-concentration), and withLNA-antimiRs SEQ ID #3227 and SEQ ID #3226; 0-50 nM end concentrations)together with 0.17 μl Lipofectamine2000 (Invitrogen) according tomanufacturer's instructions. After 24 hours, cells were harvested forluciferase measurements.

Luciferase Assay:

Growth medium was discarded and 30 μl× Passive Lysis Buffer (Promega)was added to each well. After 15-30 minutes of incubation on an orbitalshaker, renilla and firefly luciferase measurements were performedaccording to manufacturer's instructions (Promega).

Example 30. Assessment of Endogenous Let-7 Antagonism by SEQ ID #3227,an 8-Mer LNA-antimiRs, in HeLa Cells Transfected with a LuciferaseSensor Assay

In order to determine the efficiency of a fully LNA-modified 8-meroligonucleotide in targeting and antagonizing endogenous let-7, the sameluciferase sensor construct as described in previous two examples, wasco-transfected with SEQ ID #3227 into the cervical cancer cell line HeLa(that expresses moderate to high levels of let-7 as determined by Q-PCR;data not shown). Empty psiCHECK-2 vector was included as a negativecontrol.

Results:

As seen in FIG. 30, the fully LNA-modified 8-mer LNA-antimiR SEQ ID#3227 shows potent antagonism of endogenous let-7, and gives full targetde-repression at concentrations of 5-10 nM. The de-repression ofluciferase activity is dose-dependent, starting around 1 nM and reachinga plateau at approximately 10 nM.

Conclusion:

To conclude, the 8-mer LNA-antimiR (SEQ ID #3227), is a potentantagonist against also endogenous let-7 in vitro, and thus providesdefinite evidence that entire miRNA families can be successfullytargeted by short and fully LNA-modified antagonists.

Materials and Methods:

Cell Line:

The cervical cancer cell line HeLa was purchased from ATCC (#CCL-2™).HeLa cells were cultured in Eagle's MEM medium, supplemented with 10%fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

8,000 cells were seeded per well in a 96-well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HeLa cells in each well were co-transfected with 20ng HMGA2 3′UTR/psiCHECK2 plasmid or psiCHECK-2 (empty vector), and withLNA-antimiR SEQ ID #3227 (0-50 nM, end concentrations) together with0.17 μl Lipofectamine2000 (Invitrogen) according to manufacturer'sinstructions. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

Growth media was discarded and 30 μl 1× Passive Lysis Buffer (Promega)was added to each well. After 15-30 minutes of incubation on an orbitalshaker, renilla and firefly luciferase measurements were performedaccording to manufacturer's instructions (Promega).

Example 31. Assessment of miR-21 Antagonism by an 8-Mer LNA-antimiR-21(#3205) Versus an 8-Mer (#3219) Scrambled Control LNA in the Human ColonCarcinoma Cell Line HCT116

We have previously shown in this application, that an 8-mer LNA-antimiRthat is fully LNA-modified and phosphorothiolated effectivelyantagonizes miR-21 in the human cervix carcinoma cell line HeLa, thehuman breast carcinoma cell line MCF-7, the human prostate cancer cellline PC3 and human hepatocellular carcinoma HepG2 cell line. We extendedthis screening approach to the human colon carcinoma cell line HCT116.To assess the efficiency of the 8-mer LNA-antimiR oligonucleotideagainst miR-21, luciferase reporter constructs were generated in which aperfect match target site for the mature miR-21 was cloned into the3′UTR of the Renilla luciferase gene. In order to monitor miR-21inhibition, HCT116 cells were transfected with the luciferase constructstogether with the miR-21 antagonist #3205 (8-mer) and for comparison ofspecificity with the 8-mer LNA scrambled control (#3219). After 24hours, luciferase activity was measured.

Results:

The luciferase reporter experiments showed a dose-dependentde-repression of the luciferase miR-21 reporter activity with the 8-merLNA-antimiR against miR-21 (#3205) and complete de-repression wasobtained at 5 nM (FIG. 31). When comparing the specificity of the 8-merperfect match and the 8-mer scrambled control, the scrambled controlLNA-antimiR (#3219) did not show any de-repression at all, demonstratinghigh specificity of the LNA-antimiR compound against miR-21.

Conclusion:

The 8-mer (#3205) is potent in targeting miR-21 and antagonism of miR-21by #3205 is specific.

Materials and Methods:

Cell Line:

The human colon carcinoma HCT116 cell line was purchased from ATCC(CCL-247). HCT116 cells were cultured in RPMI medium, supplemented with10% fetal bovine serum, and 25 ug/ml Gentamicin.

Transfection:

110.000 cells were seeded per well in a 12-well plate and transfectionwas performed. HCT116 cells were transfected with 0.3 μg miR-21luciferase sensor plasmid or empty psiCHECK2 vector together with 1.2 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. Transfected were also varying concentrations ofLNA-antimiR and control oligonucleotides. After 24 hours, cells wereharvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 250 μl 1× Passive Lysis Buffer(Promega) was added to the wells. The plates were placed on a shaker for30 min., after which the cell lysates were transferred to eppendorftubes. The cell lysate was centrifugated for 10 min at 2.500 rpm afterwhich 50 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to the manufacturer's instructions(Promega).

Example 32. Knock-Down of miR-21 by the 8-Mer #3205 LNA-antimiR ReducesColony Formation of PC3 Cells

A hallmark of cellular transformation is the ability for tumour cells togrow in an anchorage-independent way in semisolid medium. We thereforeperformed soft agar assay which is a phenotypic assay that is relevantfor cancer, given that it measures the decrease of tumour cells. Wetransfected #3205 (perfect match LNA-antimiR-21) and #3219 (LNAscrambled control) into PC3 cells, and after 24 hours plated cells insoft agar. Colonies were counted after 12 days. We show in FIG. 32 thatinhibition of miR-21 by #3205 can reduce the amount of colonies growingin soft agar compared to the scrambled control LNA treated or untreatedcontrol (transfected, but with no LNA), demonstrating decrease of tumourcells.

Conclusion:

The 8-mer (#3205) targeting the miR-21 family reduces the number ofcolonies in soft agar, demonstrating proliferation arrest of PC3 cells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714). PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250,000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with 25 nM of different LNAoligonucleotides with Lipofectamine2000.

Clonogenic Growth in Soft Agar:

2.5×10³ PC3 cells were seeded in 0.35% agar on the top of a base layercontaining 0.5% agar. Cells were plated 24 hours after transfection.Plates were incubated in at 37° C., 5% CO₂ in a humified incubator for12 days and stained with 0.005% crystal violet for 1 h, after whichcells were counted. The assay was performed in triplicate.

Example 33. Silencing of miR-21 by the 8-Mer #3205 LNA-antimiR ReducesColony Formation of HepG2 Cells

miR-21 is overexpressed in the human hepatocellular carcinoma cell lineHepG2 and we have previously shown that we are able to regulate theluciferase activity of a miR-21 sensor plasmid with #3205 in thesecells. HepG2 cells were transfected with #3205 and #3219 (scrambled8-mer), and after 24 hours plated into soft agar. Colonies were countedafter 17 days with a microscope.

Results:

We show in FIG. 33 that inhibition of miR-21 by #3205 can reduce theamount of colonies growing in soft agar, showing that proliferationarrest has occurred. In addition, our scrambled 8-mer control, #3219,had no significant effect on the number of colonies.

Conclusion:

The 8-mer (#3205) targeting the miR-21 reduces the number of colonies insoft agar, indicating proliferation arrest of HepG2 cells.

Materials and Methods:

Cell Line:

The human hepatocytic HepG2 cell line was purchased from ECACC(#85011430). HepG2 cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

650.000 cells were seeded per well in a 6-well plate and reversetransfection was performed. HepG2 cells were transfected with 0.6 μgmiR-21 luciferase sensor plasmid or empty psiCHECK2 vector together with2.55 μl Lipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. Transfected were also LNA-antimiR and controloligonucleotides as varying concentrations. After 24 hours, the cellswere harvested for luciferase measurements.

Clonogenic Growth in Soft Agar:

2.0×10³ HepG2 cells were seeded in 0.35% agar on the top of a base layercontaining 0.5% agar. Cells were plated 24 hours after transfection.Plates were incubated in at 37° C., 5% CO₂ in a humified incubator for17 days and stained with 0.005% crystal violet for 1 h, after whichcells were counted. The assay was performed in triplicate.

Example 34. Silencing of miR-21 by the 8-Mer #3205 LNA-antimiR InhibitsCell Migration in Pc3 Cells

Cell migration can be monitored by performing a wound healing assay(=scratch assay) where a “scratch” is made in a cell monolayer, andimages are captured at the beginning and at regular intervals duringcell migration. By comparing the images, quantification of the migrationrate of the cells can be determined. This was done in the human prostatecancer cell line PC3. Cells were seeded, and on day 3 the cells weretransfected, and the next day, when 100% confluency was reached, ascratch (=wound) was made. When the scratch was made, pictures weretaken in order to document the initial wound. Afterwards the area of thewound closure is measured at different time points with the freesoftware program Image J. As shown in FIG. 34A, PC3 cells had beentreated with 25 nM #3205 (perfect match, miR-21), the control #3219 orleft untransfected. Pictures were taken after 24 hours, and the area wascalculated for the wound closure at respective time-point. The woundclosure for the untransfected cells and for the control, #3219, wasfaster as compared to our LNA-antimiR against miR-21, #3205, indicatingthat #3205 inhibits miR-21 and prevents the cells from migrating (FIG.34B).

Conclusion:

The 8-mer (#3205) targeting miR-21 inhibits the cell migration of PC3cells compared to untransfected and control transfected cells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714). PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Scratch Assay:

150.000 cells were seeded per well in a 6-well plate three days beforetransfection in order to receive 100% confluency the next day. At 24hours after transfection, a scratch was made in the cell monolayer witha 200 μl tip. Pictures were taken at 0 h and after 24 hours by using adigital camera coupled to a microscope. The software program Image J wasused to determine wound closure.

Example 35. Length Assessment of Fully LNA-Substituted LNA-antimiRsAntagonizing miR-155

We have previously shown a length assessment for miR-21 regarding fullyLNA-substituted LNA-antimiRs, and showed that the most potentLNA-antimiRs are 7-, 8- or 9 nt in length. The same experiment wasrepeated with miR-155. A perfect match target site for miR-155 wascloned into the 3′UTR of the luciferase gene in the reporter plasmidpsiCHECK2 and transfected into the mouse RAW macrophage cell linetogether with fully LNA-substituted LNA-antimiRs of different lengths.Because the endogenous levels of miR-155 are low in the RAW cell line,the cells were treated with 100 ng/ml LPS for 24 hours in order toinduce miR-155 accumulation. After 24 hours, luciferase analysis wasperformed.

Results:

As shown in FIG. 35, the most potent LNA-antimiRs are #3207(8 nt) and#3241 (9 nt), reaching almost a 80% de-repression at only 0.25 nM LNAconcentration. The 6-mer (#3244) shows no significant de-repression.Increasing the length to 12-mer to 14-mer (#3242 and #3243) decreasedthe potency as shown by less efficient de-repression of the miR-155reporter.

Conclusion:

The most potent fully LNA-substituted LNA-antimiRs targeting miR-155were an 8- and 9-mer (#3207 and #3241).

Materials and Methods:

Cell Line:

The mouse macrophage RAW 264.7 cell line was purchased from ATCC(TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10%fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500,000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, RAW 264.7 cells were transfected with 0.3 ug miR-155perfect match/psiCHECK2 or empty psiCHECK2 vector together with 10 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. Transfected was also varying concentrations ofLNA-antimiRs. In order to induce miR-155 accumulation, LPS (100 ng/ml)was added to the RAW cells after the 4 hour incubation with thetransfection complexes. After another 24 hours, cells were harvested forluciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, afterwhich cells were spinned for 5 min at 2.500 rpm. The supernatant wasdiscarded and 50 μl× Passive Lysis Buffer (Promega) was added to thecell pellet, after which cells were put on ice for 30 min. The lysedcells were spinned at 10.000 rpm for 30 min after which 20 μl weretransferred to a 96-well plate and luciferase measurements wereperformed according to the manufacturer's instructions (Promega).

Example 36. Plasma Protein Binding for the Fully LNA-Substituted 8-Mer#3205 Targeting miR-21 (LNA-antimiR-21)

The plasma proteins are not saturated with #3205 at the plasmaconcentrations in the experiment shown in FIG. 36A. In a wide range of#3205 concentrations in the plasma the protein binding is around 95% ofthe #3205 LNA-antimiR-21 in FIG. 36B. At #3205 concentrations 50.1 μM(174 μg/mL) the binding capacity of plasma proteins for FAM-labeled#3205 has not been saturated.

Materials and Methods:

Mouse plasma (100 μL) was spiked with FAM-labeled #3205 to 0.167, 1.67,5.01, 10.02, 16.7, 25.05 and 50.1 μM concentrations. The solutions wereincubated at 37° C. for 30 minutes. The solutions were transferred to aMicrocon Ultracel YM-30 filter (regenerated cellulose 30.000 MWCO). Thefilters were spun for 20 minutes at 2000 g and at room temperature in amicrocentrifuge. The filtrate was diluted 5, 10 and 20 times and 100 μLsamples were transferred to a microtiter plate (Polystyrene BlackNUNC-237108). The fluorescence was detected using a FLUOstar Optimaelisa reader with excitation 458 nm and emission 520 nm. The amount ofunbound FAM-labeled #3205 was calculated from a standard curve derivedfrom filtrated plasma spiked with FAM-labeled #3205 at 12 different(0.45-1000 nM) concentrations. The numbers were corrected with therecovery number established from filtration experiments with #3205concentrations 0.167, 1.67, 5.01, 10.02, 16.7, 25.05 and 50.1 μM infiltrated plasma. The recovery of FAM-labeled #3205 was 86%.

Example 37. Quantitative Whole Body Autoradiography Study in FemalePigmented Mice after Single Intravenous Administration of ³⁵S-Labelled#3205 LNA-antimiR-21

In order to determine the biodistribution of a short fully LNA-modifiedLNA-antimiR (#3205, 8-mer) a whole body tissue distribution ofradioactively labeled compound was done in mice. ³⁵S-labelled #3205 wasdosed to mice with a single intravenous administration and mice weresacrificed at different time-points, ranging from 5 min to 21 days.

TABLE 6(i) Individual tissue concentrations (μg #3205/g tissue) after asingle intravenous administration of ³⁵S- labelled #3205 in femalepigmented mice. The figures are mean values of three measurements foreach tissue and ratio. The coefficient of variation (CV) is generallyabout 10%. Max. Conc. of oligo μg Time of max Tissue #3205/g tissueconc. hours T½ hours Adrenal gl. 13.6 0.083 374 Bile 4 1 Bone marrow 7.20.083 411 Brain 0.4 0.083 Brown fat 8.8 0.083 Gastric muc. 10.1 0.083Heart blood 26.2 0.083 10.3 Kidney ctx. 58.7 24 104 Liver 11.8 0.083 58810.7 24 Lung 13.2 0.083 289 Lymph node 5 0.083 262 2.4 48 Lymph 18.8 420.8 168 Myocardium 8.1 0.083 662 Ovary 13 0.083 198 Pancreas 5 0.083Pituitary gl. 6.7 0.083 Salivary gl. 8.6 0.083 405 5.5 168 skel. Muscle4.8 0.083 Skin pig. 5.4 0.25 Spleen 9.8 0.083 564 Thymus 3.8 0.083 185Thyroid gl. 10.9 0.083 592 Urine 328.9 0.083 Uterus 9.6 0.25 177 Uvea ofthe eye 13.6 0.083 LOQ 0.045 0.083 0.033 24 0.03 168

TABLE 6(ii) Tissue to liver ratios after single intravenousadministration of ³⁵S-labelled #3205 in female pigmented mice. Animal no³⁵S-#3205 10 11 12 13 14 15 16 17 18 Surv. Time (h) 0.083 0.25 1 h 4 h24 h 48 h 96 h 168 504 Organ Adrenal gl liver liver liver liver liverliver liver liver liver Bile 1.15 1.08 0.52 0.27 0.24 0.26 0.23 0.180.17 Bone marrow 0.03 0.11 0.55 0.10 0.03 0.07 0.04 0.03 0.04 Brain 0.610.81 0.55 0.45 0.40 0.48 0.43 0.42 0.34 Brown fat 0.03 0.03 0.01 0.000.00 0.00 0.00 0.00 0.00 Gastric muc 0.75 0.57 0.29 0.12 0.07 0.12 0.080.10 0.07 Heart blood 0.86 0.71 0.31 0.22 0.10 0.21 0.15 0.16 0.12Kidney ctx 2.23 1.91 0.74 0.11 0.01 0.00 0.00 0.00 0.00 Liver 2.87 3.946.45 6.95 5.51 6.68 3.92 2.24 0.40 Lung 1.00 1.00 1.00 1.00 1.00 1.001.00 1.00 1.00 Lymph node 1.12 0.97 0.63 0.09 0.04 0.04 0.03 0.02 0.02Lymph 0.43 0.30 0.25 0.19 0.11 0.32 0.20 0.17 0.12 Myocardium 0.82 1.091.78 2.78 1.03 2.05 1.62 3.17 1.89 Ovary 0.69 0.63 0.30 0.13 0.10 0.150.09 0.11 0.12 Pancreas 1.10 1.40 0.61 0.31 0.27 0.28 0.21 0.21 0.08Pituitary gland 0.42 0.37 0.22 0.18 0.12 0.17 0.12 0.15 0.11 Salivarygland 0.57 0.54 0.28 0.11 0.15 0.16 0.12 0.10 0.08 Skel. muscle 0.730.81 0.38 0.25 0.25 0.42 0.23 0.85 0.24 Skin. pigm. 0.40 0.28 0.14 0.040.02 0.04 0.03 0.03 0.03 Spleen 0.34 0.69 0.65 0.36 0.20 0.26 0.20 0.190.13 Thymus 0.83 0.86 0.44 0.32 0.24 0.34 0.35 0.29 0.31 Thyroid gland0.32 0.31 0.14 0.07 0.09 0.08 0.05 0.04 0.02 Urine 0.9 1.2 0.43 0.280.25 0.34 0.19 0.26 0.25 Uterus 27.96 39.48 9.90 5.44 0.24 0.39 0.120.15 0.03 Uvea of the eye 0.56 1.23 0.65 0.30 0.30 0.07 0.27 0.16 0.08Conclusions:

#3205 shows blood clearance of radioactivity with elimination half-livesof 8-10 hours. High levels of radioactivity were registered in thekidney cortex, lymph, liver, bone marrow, spleen, ovary and uterus. Thehighest level of radioactivity was registered in the kidney cortexshowing five times higher levels than that of the liver for #3205. Astrong retention of radioactivity was noticed in the kidney cortex,lymph, liver, bone marrow and spleen for #3205 LNA-antimiR-21.

Materials and Methods:

Dose Administration:

All mice were weighed before administration. Nine female mice were given10 mg/kg of ³⁵S-#3205 intravenously in a tail vein. The volume given toeach animal was 10 mL/kg of the test formulation. The specific activity75.7 μCi/mg. Individual mice were killed 5 min, 15 min, 1 hour, 4 hours,24 hours, 2 days, 4 days, 7 days and 21 days after administration of#3205. Whole body autoradiography: The mice were anaesthetized bysevoflurane, and then immediately immersed in heptane, cooled with dryice to −80° C., ABR-SOP-0130. The frozen carcasses were embedded in agel of aqueous carboxymethyl cellulose (CMC), frozen in ethanol, cooledwith dry ice (−80° C.) and sectioned sagittaly for whole bodyautoradiography, according to the standard method, ABR-SOP-0131. Fromeach animal 20 μm sections were cut at different levels with acryomicrotome (Leica CM 3600) at a temperature of about −20° C. Theobtained sections were caught on tape (Minnesota Mining andManufacturing Co., No. 810) and numbered consecutively with radioactiveink. After being freeze-dried at −20° C. for about 24 hours, selectedsections were covered with a thin layer of mylar foil, and put onimaging plates (Fuji, Japan). Exposure took place in light tightcassettes in a lead shielding box at −20° C., to protect the imageplates from environmental radiation. After exposure the imaging plateswere scanned at a pixel size of 50 μm and analyzed by radioluminographyusing a bioimaging analysis system (Bas 2500, Fuji, Japan), anddescribed in ABR-SOP-0214. A water-soluble standard test solution of ³⁵Sradioactivity was mixed with whole blood and used for production of acalibration scale, ABR-SOP-0251. However, the different blood standardswere dissolved in 500 uL Soluene-35. 4.5 mL Ultima Gold was then addedto the dissolved samples. As 35S and ¹⁴C have very similar energyspectra, a standard ¹⁴C-programme (Packard 2200CA) was used when theradioactivity for the different blood samples was settled.

Pharmacokinetic Calculations:

The 35S radioactivity measured in whole blood and tissues was expressedas nCi/g tissue and recalculated to nmol equiv/g tissue for thepharmacokinetic evaluation. The pharmacokinetic parameters C_(max),t_(1/2) and AUC were determined for the whole blood and tissues bynon-compartmental analysis using WinNonlin Professional (PharsightCorporation, Mountain View, Calif., USA). After intravenousadministration, the concentration was extrapolated back to zero andexpressed as (C₀). The elimination rate constant λ was estimated bylinear regression analysis of the terminal slope of the logarithmicplasma concentration-time curve. The elimination half-life, t_(1/2), wascalculated using the equation, t_(1/2)=ln 2/λ. The last threetime-points above LOQ were used in the elimination half-lifecalculations, if not stated otherwise.

Example 38. Assessment of Let-7 Inhibition In Vivo by an 8-MerLNA-antimiR, as Determined Through Ras Protein Quantification in MouseLung and Kidney

In order to investigate the possibility to antagonize the abundantlyexpressed let-7 family in vivo, mice were intravenously (i.v.) injectedwith an 8-mer LNA-antimiR antagonist or with saline. To measuretreatment effect, proteins were isolated from lungs and kidneys. Becausethe Ras family of proteins (N-Ras, K-Ras, and H-Ras), in particularN-Ras and K-Ras, has previously been shown to be regulated (repressed)by the let-7 family by Johnson et al. (Cell, 2005), the aim was toanalyze whether these let-7 targets could be de-repressed in vivo.

Results:

As seen in FIG. 37, the 8-mer LNA-antimiR potently de-repressed Rasprotein levels in the kidneys of treated mice, normalized against salinecontrols. The up-regulation in this organ was more than 3-fold, showinga clear in vive effect. In the lungs, however, only a minimal (1.2-fold)Ras de-repression was observed (FIG. 1B), suggesting that insufficientamounts of LNA-antimiR has entered this organ in order to inhibit itsmassive amounts of let-7, as previously described by Johnson et al.(Cancer Research, 2007).

Conclusion:

The 8-mer LNA-antimiR shows a clear effect in regulating target let-7miRNA in vivo, as evaluated based on Ras protein levels in treated vs.control mice. Whereas the effect seems to be smaller in lungs, Raslevels in the kidney show a substantial up-regulation uponantimiRs-treatment.

Materials and Methods:

Animals and Dosing:

C57BL/6 female mice were treated with 10 mg/kg LNA-antimiR or saline forthree consecutive days (0, 1, and 2) and sacrificed on day 4. Tissuesamples from lungs and kidneys were snapfrozen and stored at −80° C.until further processing.

Western Blot Analysis:

Lung and kidney proteins from saline and LNA-antimiR-treated mice wereseparated on NuPAGE Bis Tris 4-12% (Invitrogen), using 100 μg persample. The proteins were transferred to a nitrocellulose membrane usingiBlot (Invitrogen) according to the manufacturer's instructions.Blocking, antibody dilution and detection was performed according to themanufacturer's specifications. For Ras detection, a primary rabbit-antiRas antibody (SC-3339, Santa Cruz Biotechnology) and a secondaryHRP-conjugated swine-anti-rabbit antibody (P0399, Dako) was used, andfor tubulin detection, a primary tubulin alpha (MS-581-P1, Neomarkers)and a secondary HRP-conjugated goat-anti-mouse antibody (P0447, Dako)was used.

Example 40. In Vivo Efficacy Assessment of the 8-Mer LNA-antimiR (#3205)in Targeting miR-21, as Determined by Pdcd4 Protein Up-Regulation inMouse Kidney

We have shown that an 8-mer LNA-antimiR that is fully LNA-modifiedantagonizes miR-21 and has the ability to regulate the protein levels ofthe miR-21 target Pdcd4 in vitro. We therefore injected the LNA-antimiRinto mice to determine the effects of the LNA-antimiR in vivo. The micereceived 25 mg/kg of #3205 by i.v. injection every other day for 14 days(a total of 5 doses). The mice were sacrificed on day 14, the kidney wasremoved, and protein was isolated. In order to determine targetregulation, Western blot analysis was performed.

Results:

As shown in FIG. 38, treating mice with #3205 showed significantlyincreased Pdcd4 protein levels as compared to the saline control. Whilethe normalized Pdcd4 versus Gapdh ratio was consistent in both salinesamples, the protein up-regulation in the two LNA-antimiR-treated(#32059 mice were measured to 3.3- and 6.3-fold, respectively,demonstrating an in vivo pharmacological effect of the #3205 8-merLNA-antimiR.

Conclusion:

The fully LNA-modified 8-mer LNA-antimiR #3205 antagonizes miR-21 invivo, as demonstrated through its ability to de-repress (up-regulate)mouse kidney levels of Pdcd4, a validated miR-21 target.

Materials and Methods:

Animals and Dosing:

C57BL/6 female mice with average of 20 g body weight at first dosingwere used in all experiments and received regular chow diet (Altromin no1324, Brogaarden, Gentofte, Denmark). Substances were formulated inphysiological saline (0.9% NaCl). The animals were dozed withLNA-antimiR or saline (0.9% NaCl), receiving an injection of 25 mg/kgevery other day for 14 days, a total of 5 doses. Animals were sacrificedon day 14.

Western Blot Analysis:

80 μg kidney tissue from saline or LNA-treated mice was separated onNuPAGE Bis Tris 4-12% (Invitrogen). The proteins were transferred to anitrocellulose membrane using iBlot (Invitrogen) according to themanufacturer's instructions. The membrane was incubated with Pdcd4antibody (Bethyl Laboratories), followed by HRP-conjugatedswine-anti-rabbit antibody (Dako). As equal loading control, GAPDH(Abcam) was used, followed by HRP-conjugated swine-anti-mouse antibody.The membranes were visualized by chemiluminiscence (ECL, Amersham).

TABLE 1 SEQ SEQ SEQ SEQ ID ID ID ID microRNA MicroRNASequence NO 9-merNO 8-mer NO 7-mer NO ebv-miR-BART1-3p UAGCACCGCUAUCCACUAUGUC  40AGCGGTGCT  977 GCGGTGCT 1914 CGGTGCT 2851 ebv-miR-BART1-5pUCUUAGUGGAAGUGACGUGCUGUG  41 TCCACTAAG  978 CCACTAAG 1915 CACTAAG 2852ebv-miR-BART10 UACAUAACCAUGGAGUUGGCUGU  42 TGGTTATGT  979 GGTTATGT 1916GTTATGT 2853 ebv-miR-BART10* GCCACCUCUUUGGUUCUGUACA  43 AAGAGGTGG  980AGAGGTGG 1917 GAGGTGG 2854 ebv-miR-BART11-3p ACGCACACCAGGCUGACUGCC  44TGGTGTGCG  981 GGTGTGCG 1918 GTGTGCG 2855 ebv-miR-BART11-5pUCAGACAGUUUGGUGCGCUAGUUG  45 AACTGTCTG  982 ACTGTCTG 1919 CTGTCTG 2856ebv-miR-BART12 UCCUGUGGUGUUUGGUGUGGUU  46 CACCACAGG  983 ACCACAGG 1920CCACAGG 2857 ebv-miR-BART13 UGUAACUUGCCAGGGACGGCUGA  47 GCAAGTTAC  984CAAGTTAC 1921 AAGTTAC 2858 ebv-miR-BART13* AACCGGCUCGUGGCUCGUACAG  48CGAGCCGGT  985 GAGCCGGT 1922 AGCCGGT 2859 ebv-miR-BART14UAAAUGCUGCAGUAGUAGGGAU  49 GCAGCATTT  986 CAGCATTT 1923 AGCATTT 2860ebv-miR-BART14* UACCCUACGCUGCCGAUUUACA  50 GCGTAGGGT  987 CGTAGGGT 1924GTAGGGT 2861 ebv-miR-BART15 GUCAGUGGUUUUGUUUCCUUGA  51 AACCACTGA  988ACCACTGA 1925 CCACTGA 2862 ebv-miR-BART16 UUAGAUAGAGUGGGUGUGUGCUCU  52CTCTATCTA  989 TCTATCTA 1926 CTATCTA 2863 ebv-miR-BART17-3pUGUAUGCCUGGUGUCCCCUUAGU  53 CAGGCATAC  990 AGGCATAC 1927 GGCATAC 2864ebv-miR-BART17-5p UAAGAGGACGCAGGCAUACAAG  54 CGTCCTCTT  991 GTCCTCTT1928 TCCTCTT 2865 ebv-miR-BART18-3p UAUCGGAAGUUUGGGCUUCGUC  55 ACTTCCGAT 992 CTTCCGAT 1929 TTCCGAT 2866 ebv-miR-BART18-5p UCAAGUUCGCACUUCCUAUACA 56 GCGAACTTG  993 CGAACTTG 1930 GAACTTG 2867 ebv-miR-BART19-3pUUUUGUUUGCUUGGGAAUGCU  57 GCAAACAAA  994 CAAACAAA 1931 AAACAAA 2868ebv-miR-BART19-5p ACAUUCCCCGCAAACAUGACAUG  58 CGGGGAATG  995 GGGGAATG1932 GGGAATG 2869 ebv-miR-BART2-3p AAGGAGCGAUUUGGAGAAAAUAAA  59ATCGCTCCT  996 TCGCTCCT 1933 CGCTCCT 2870 ebv-miR-BART2-5pUAUUUUCUGCAUUCGCCCUUGC  60 GCAGAAAAT  997 CAGAAAAT 1934 AGAAAAT 2871ebv-miR-BART20-3p CAUGAAGGCACAGCCUGUUACC  61 TGCCTTCAT  998 GCCTTCAT1935 CCTTCAT 2872 ebv-miR-BART20-5p UAGCAGGCAUGUCUUCAUUCC  62 ATGCCTGCT 999 TGCCTGCT 1936 GCCTGCT 2873 ebv-miR-BART3 CGCACCACUAGUCACCAGGUGU  63TAGTGGTGC 1000 AGTGGTGC 1937 GTGGTGC 2874 ebv-miR-BART3*ACCUAGUGUUAGUGUUGUGCU  64 AACACTAGG 1001 ACACTAGG 1938 CACTAGG 2875ebv-miR-BART4 GACCUGAUGCUGCUGGUGUGCU  65 GCATCAGGT 1002 CATCAGGT 1939ATCAGGT 2876 ebv-miR-BART5 CAAGGUGAAUAUAGCUGCCCAUCG  66 ATTCACCTT 1003TTCACCTT 1940 TCACCTT 2877 ebv-miR-BART6-3p CGGGGAUCGGACUAGCCUUAGA  67CCGATCCCC 1004 CGATCCCC 1941 GATCCCC 2878 ebv-miR-BART6-5pUAAGGUUGGUCCAAUCCAUAGG  68 ACCAACCTT 1005 CCAACCTT 1942 CAACCTT 2879ebv-miR-BART7 CAUCAUAGUCCAGUGUCCAGGG  69 GACTATGAT 1006 ACTATGAT 1943CTATGAT 2880 ebv-miR-BART7* CCUGGACCUUGACUAUGAAACA  70 AAGGTCCAG 1007AGGTCCAG 1944 GGTCCAG 2881 ebv-miR-BART8 UACGGUUUCCUAGAUUGUACAG  71GGAAACCGT 1008 GAAACCGT 1945 AAACCGT 2882 ebv-miR-BART8*GUCACAAUCUAUGGGGUCGUAGA  72 AGATTGTGA 1009 GATTGTGA 1946 ATTGTGA 2883ebv-miR-BART9 UAACACUUCAUGGGUCCCGUAGU  73 TGAAGTGTT 1010 GAAGTGTT 1947AAGTGTT 2884 ebv-miR-BART9* UACUGGACCCUGAAUUGGAAAC  74 GGGTCCAGT 1011GGTCCAGT 1948 GTCCAGT 2885 ebv-miR-BHRF1-1 UAACCUGAUCAGCCCCGGAGUU  75GATCAGGTT 1012 ATCAGGTT 1949 TCAGGTT 2886 ebv-miR-BHRF1-2UAUCUUUUGCGGCAGAAAUUGA  76 GCAAAAGAT 1013 CAAAAGAT 1950 AAAAGAT 2887ebv-miR-BHRF1-2* AAAUUCUGUUGCAGCAGAUAGC  77 AACAGAATT 1014 ACAGAATT 1951CAGAATT 2888 ebv-miR-BHRF1-3 UAACGGGAAGUGUGUAAGCACA  78 CTTCCCGTT 1015TTCCCGTT 1952 TCCCGTT 2889 hcmv-miR-UL112 AAGUGACGGUGAGAUCCAGGCU  79ACCGTCACT 1016 CCGTCACT 1953 CGTCACT 2890 hcmv-miR-UL148DUCGUCCUCCCCUUCUUCACCG  80 GGGAGGACG 1017 GGAGGACG 1954 GAGGACG 2891hcmv-miR-UL22A UAACUAGCCUUCCCGUGAGA  81 AGGCTAGTT 1018 GGCTAGTT 1955GCTAGTT 2892 hcmv-miR-UL22A* UCACCAGAAUGCUAGUUUGUAG  82 ATTCTGGTG 1019TTCTGGTG 1956 TCTGGTG 2893 hcmv-miR-UL36 UCGUUGAAGACACCUGGAAAGA  83TCTTCAACG 1020 CTTCAACG 1957 TTCAACG 2894 hcmv-miR-UL36*UUUCCAGGUGUUUUCAACGUGC  84 CACCTGGAA 1021 ACCTGGAA 1958 CCTGGAA 2895hcmv-miR-UL70-3p GGGGAUGGGCUGGCGCGCGG  85 GCCCATCCC 1022 CCCATCCC 1959CCATCCC 2896 hcmv-miR-UL70-5p UGCGUCUCGGCCUCGUCCAGA  86 CCGAGACGC 1023CGAGACGC 1960 GAGACGC 2897 hcmv-miR-US25-1 AACCGCUCAGUGGCUCGGACC  87CTGAGCGGT 1024 TGAGCGGT 1961 GAGCGGT 2898 hcmv-miR-US25-1*UCCGAACGCUAGGUCGGUUCUC  88 AGCGTTCGG 1025 GCGTTCGG 1962 CGTTCGG 2899hcmv-miR-US25-2- AUCCACUUGGAGAGCUCCCGCGG  89 CCAAGTGGA 1026 CAAGTGGA1963 AAGTGGA 2900 3p hcmv-miR-US25-2- AGCGGUCUGUUCAGGUGGAUGA  90ACAGACCGC 1027 CAGACCGC 1964 AGACCGC 2901 5p hcmv-miR-US33-3pUCACGGUCCGAGCACAUCCA  91 CGGACCGTG 1028 GGACCGTG 1965 GACCGTG 2902hcmv-miR-US33-5p GAUUGUGCCCGGACCGUGGGCG  92 GGGCACAAT 1029 GGCACAAT 1966GCACAAT 2903 hcmv-miR-US4 CGACAUGGACGUGCAGGGGGAU  93 GTCCATGTC 1030TCCATGTC 1967 CCATGTC 2904 hcmv-miR-US5-1 UGACAAGCCUGACGAGAGCGU  94AGGCTTGTC 1031 GGCTTGTC 1968 GCTTGTC 2905 hcmv-miR-US5-2UUAUGAUAGGUGUGACGAUGUC  95 CCTATCATA 1032 CTATCATA 1969 TATCATA 2906hsa-let-7a UGAGGUAGUAGGUUGUAUAGUU  96 TACTACCTC 1033 ACTACCTC 1970CTACCTC 2907 hsa-let-7a* CUAUACAAUCUACUGUCUUUC  97 GATTGTATA 1034ATTGTATA 1971 TTGTATA 2908 hsa-let-7b UGAGGUAGUAGGUUGUGUGGUU  98TACTACCTC 1035 ACTACCTC 1972 CTACCTC 2909 hsa-let-7b*CUAUACAACCUACUGCCUUCCC  99 GGTTGTATA 1036 GTTGTATA 1973 TTGTATA 2910hsa-let-7c UGAGGUAGUAGGUUGUAUGGUU 100 TACTACCTC 1037 ACTACCTC 1974CTACCTC 2911 hsa-let-7c* UAGAGUUACACCCUGGGAGUUA 101 TGTAACTCT 1038GTAACTCT 1975 TAACTCT 2912 hsa-let-7d AGAGGUAGUAGGUUGCAUAGUU 102TACTACCTC 1039 ACTACCTC 1976 CTACCTC 2913 hsa-let-7d*CUAUACGACCUGCUGCCUUUCU 103 GGTCGTATA 1040 GTCGTATA 1977 TCGTATA 2914hsa-let-7e UGAGGUAGGAGGUUGUAUAGUU 104 TCCTACCTC 1041 CCTACCTC 1978CTACCTC 2915 hsa-let-7e* CUAUACGGCCUCCUAGCUUUCC 105 GGCCGTATA 1042GCCGTATA 1979 CCGTATA 2916 hsa-let-7f UGAGGUAGUAGAUUGUAUAGUU 106TACTACCTC 1043 ACTACCTC 1980 CTACCTC 2917 hsa-let-7f-1*CUAUACAAUCUAUUGCCUUCCC 107 GATTGTATA 1044 ATTGTATA 1981 TTGTATA 2918hsa-let-7f-2* CUAUACAGUCUACUGUCUUUCC 108 GACTGTATA 1045 ACTGTATA 1982CTGTATA 2919 hsa-let-7g UGAGGUAGUAGUUUGUACAGUU 109 TACTACCTC 1046ACTACCTC 1983 CTACCTC 2920 hsa-let-7g* CUGUACAGGCCACUGCCUUGC 110GCCTGTACA 1047 CCTGTACA 1984 CTGTACA 2921 hsa-let-7iUGAGGUAGUAGUUUGUGCUGUU 111 TACTACCTC 1048 ACTACCTC 1985 CTACCTC 2922hsa-let-7i* CUGCGCAAGCUACUGCCUUGCU 112 GCTTGCGCA 1049 CTTGCGCA 1986TTGCGCA 2923 hsa-miR-1 UGGAAUGUAAAGAAGUAUGUAU 113 TTACATTCC 1050TACATTCC 1987 ACATTCC 2924 hsa-miR-100 AACCCGUAGAUCCGAACUUGUG 114TCTACGGGT 1051 CTACGGGT 1988 TACGGGT 2925 hsa-miR-100*CAAGCUUGUAUCUAUAGGUAUG 115 TACAAGCTT 1052 ACAAGCTT 1989 CAAGCTT 2926hsa-miR-101 UACAGUACUGUGAUAACUGAA 116 CAGTACTGT 1053 AGTACTGT 1990GTACTGT 2927 hsa-miR-101* CAGUUAUCACAGUGCUGAUGCU 117 GTGATAACT 1054TGATAACT 1991 GATAACT 2928 hsa-miR-103 AGCAGCAUUGUACAGGGCUAUGA 118CAATGCTGC 1055 AATGCTGC 1992 ATGCTGC 2929 hsa-miR-103-asUCAUAGCCCUGUACAAUGCUGCU 119 AGGGCTATG 1056 GGGCTATG 1993 GGCTATG 2930hsa-miR-105 UCAAAUGCUCAGACUCCUGUGGU 120 GAGCATTTG 1057 AGCATTTG 1994GCATTTG 2931 hsa-miR-105* ACGGAUGUUUGAGCAUGUGCUA 121 AAACATCCG 1058AACATCCG 1995 ACATCCG 2932 hsa-miR-106a AAAAGUGCUUACAGUGCAGGUAG 122AAGCACTTT 1059 AGCACTTT 1996 GCACTTT 2933 hsa-miR-106a*CUGCAAUGUAAGCACUUCUUAC 123 TACATTGCA 1060 ACATTGCA 1997 CATTGCA 2934hsa-miR-106b UAAAGUGCUGACAGUGCAGAU 124 CAGCACTTT 1061 AGCACTTT 1998GCACTTT 2935 hsa-miR-106b* CCGCACUGUGGGUACUUGCUGC 125 CACAGTGCG 1062ACAGTGCG 1999 CAGTGCG 2936 hsa-miR-107 AGCAGCAUUGUACAGGGCUAUCA 126CAATGCTGC 1063 AATGCTGC 2000 ATGCTGC 2937 hsa-miR-10aUACCCUGUAGAUCCGAAUUUGUG 127 CTACAGGGT 1064 TACAGGGT 2001 ACAGGGT 2938hsa-miR-10a* CAAAUUCGUAUCUAGGGGAAUA 128 TACGAATTT 1065 ACGAATTT 2002CGAATTT 2939 hsa-miR-10b UACCCUGUAGAACCGAAUUUGUG 129 CTACAGGGT 1066TACAGGGT 2003 ACAGGGT 2940 hsa-miR-10b* ACAGAUUCGAUUCUAGGGGAAU 130TCGAATCTG 1067 CGAATCTG 2004 GAATCTG 2941 hsa-miR-1178UUGCUCACUGUUCUUCCCUAG 131 CAGTGAGCA 1068 AGTGAGCA 2005 GTGAGCA 2942hsa-miR-1179 AAGCAUUCUUUCAUUGGUUGG 132 AAGAATGCT 1069 AGAATGCT 2006GAATGCT 2943 hsa-miR-1180 UUUCCGGCUCGCGUGGGUGUGU 133 GAGCCGGAA 1070AGCCGGAA 2007 GCCGGAA 2944 hsa-miR-1181 CCGUCGCCGCCACCCGAGCCG 134GCGGCGACG 1071 CGGCGACG 2008 GGCGACG 2945 hsa-miR-1182GAGGGUCUUGGGAGGGAUGUGAC 135 CAAGACCCT 1072 AAGACCCT 2009 AGACCCT 2946hsa-miR-1183 CACUGUAGGUGAUGGUGAGAGUGGGCA 136 ACCTACAGT 1073 CCTACAGT2010 CTACAGT 2947 hsa-miR-1184 CCUGCAGCGACUUGAUGGCUUCC 137 TCGCTGCAG1074 CGCTGCAG 2011 GCTGCAG 2948 hsa-miR-1185 AGAGGAUACCCUUUGUAUGUU 138GGTATCCTC 1075 GTATCCTC 2012 TATCCTC 2949 hsa-miR-1197UAGGACACAUGGUCUACUUCU 139 ATGTGTCCT 1076 TGTGTCCT 2013 GTGTCCT 2950hsa-miR-1200 CUCCUGAGCCAUUCUGAGCCUC 140 GGCTCAGGA 1077 GCTCAGGA 2014CTCAGGA 2951 hsa-miR-1201 AGCCUGAUUAAACACAUGCUCUGA 141 TAATCAGGC 1078AATCAGGC 2015 ATCAGGC 2952 hsa-miR-1202 GUGCCAGCUGCAGUGGGGGAG 142CAGCTGGCA 1079 AGCTGGCA 2016 GCTGGCA 2953 hsa-miR-1203CCCGGAGCCAGGAUGCAGCUC 143 TGGCTCCGG 1080 GGCTCCGG 2017 GCTCCGG 2954hsa-miR-1204 UCGUGGCCUGGUCUCCAUUAU 144 CAGGCCACG 1081 AGGCCACG 2018GGCCACG 2955 hsa-miR-1205 UCUGCAGGGUUUGCUUUGAG 145 ACCCTGCAG 1082CCCTGCAG 2019 CCTGCAG 2956 hsa-miR-1206 UGUUCAUGUAGAUGUUUAAGC 146TACATGAAC 1083 ACATGAAC 2020 CATGAAC 2957 hsa-miR-1207-3pUCAGCUGGCCCUCAUUUC 147 GGCCAGCTG 1084 GCCAGCTG 2021 CCAGCTG 2958hsa-miR-1207-5p UGGCAGGGAGGCUGGGAGGGG 148 CTCCCTGCC 1085 TCCCTGCC 2022CCCTGCC 2959 hsa-miR-1208 UCACUGUUCAGACAGGCGGA 149 TGAACAGTG 1086GAACAGTG 2023 AACAGTG 2960 hsa-miR-122 UGGAGUGUGACAAUGGUGUUUG 150TCACACTCC 1087 CACACTCC 2024 ACACTCC 2961 hsa-miR-122*AACGCCAUUAUCACACUAAAUA 151 TAATGGCGT 1088 AATGGCGT 2025 ATGGCGT 2962hsa-miR-1224-3p CCCCACCUCCUCUCUCCUCAG 152 GGAGGTGGG 1089 GAGGTGGG 2026AGGTGGG 2963 hsa-miR-1224-5p GUGAGGACUCGGGAGGUGG 153 GAGTCCTCA 1090AGTCCTCA 2027 GTCCTCA 2964 hsa-miR-1225-3p UGAGCCCCUGUGCCGCCCCCAG 154CAGGGGCTC 1091 AGGGGCTC 2028 GGGGCTC 2965 hsa-miR-1225-5pGUGGGUACGGCCCAGUGGGGGG 155 CCGTACCCA 1092 CGTACCCA 2029 GTACCCA 2966hsa-miR-1226 UCACCAGCCCUGUGUUCCCUAG 156 GGGCTGGTG 1093 GGCTGGTG 2030GCTGGTG 2967 hsa-miR-1226* GUGAGGGCAUGCAGGCCUGGAUGGGG 157 ATGCCCTCA 1094TGCCCTCA 2031 GCCCTCA 2968 hsa-miR-1227 CGUGCCACCCUUUUCCCCAG 158GGGTGGCAC 1095 GGTGGCAC 2032 GTGGCAC 2969 hsa-miR-1228UCACACCUGCCUCGCCCCCC 159 GCAGGTGTG 1096 CAGGTGTG 2033 AGGTGTG 2970hsa-miR-1228* GUGGGCGGGGGCAGGUGUGUG 160 CCCCGCCCA 1097 CCCGCCCA 2034CCGCCCA 2971 hsa-miR-1229 CUCUCACCACUGCCCUCCCACAG 161 GTGGTGAGA 1098TGGTGAGA 2035 GGTGAGA 2972 hsa-miR-1231 GUGUCUGGGCGGACAGCUGC 162GCCCAGACA 1099 CCCAGACA 2036 CCAGACA 2973 hsa-miR-1233UGAGCCCUGUCCUCCCGCAG 163 ACAGGGCTC 1100 CAGGGCTC 2037 AGGGCTC 2974hsa-miR-1234 UCGGCCUGACCACCCACCCCAC 164 GTCAGGCCG 1101 TCAGGCCG 2038CAGGCCG 2975 hsa-miR-1236 CCUCUUCCCCUUGUCUCUCCAG 165 GGGGAAGAG 1102GGGAAGAG 2039 GGAAGAG 2976 hsa-miR-1237 UCCUUCUGCUCCGUCCCCCAG 166AGCAGAAGG 1103 GCAGAAGG 2040 CAGAAGG 2977 hsa-miR-1238CUUCCUCGUCUGUCUGCCCC 167 GACGAGGAA 1104 ACGAGGAA 2041 CGAGGAA 2978hsa-miR-124 UAAGGCACGCGGUGAAUGCC 168 GCGTGCCTT 1105 CGTGCCTT 2042GTGCCTT 2979 hsa-miR-124* CGUGUUCACAGCGGACCUUGAU 169 TGTGAACAC 1106GTGAACAC 2043 TGAACAC 2980 hsa-miR-1243 AACUGGAUCAAUUAUAGGAGUG 170TGATCCAGT 1107 GATCCAGT 2044 ATCCAGT 2981 hsa-miR-1244AAGUAGUUGGUUUGUAUGAGAUGGUU 171 CCAACTACT 1108 CAACTACT 2045 AACTACT 2982hsa-miR-1245 AAGUGAUCUAAAGGCCUACAU 172 TAGATCACT 1109 AGATCACT 2046GATCACT 2983 hsa-miR-1246 AAUGGAUUUUUGGAGCAGG 173 AAAATCCAT 1110AAATCCAT 2047 AATCCAT 2984 hsa-miR-1247 ACCCGUCCCGUUCGUCCCCGGA 174CGGGACGGG 1111 GGGACGGG 2048 GGACGGG 2985 hsa-miR-1248ACCUUCUUGUAUAAGCACUGUGCUAAA 175 ACAAGAAGG 1112 CAAGAAGG 2049 AAGAAGG2986 hsa-miR-1249 ACGCCCUUCCCCCCCUUCUUCA 176 GGAAGGGCG 1113 GAAGGGCG2050 AAGGGCG 2987 hsa-miR-1250 ACGGUGCUGGAUGUGGCCUUU 177 CCAGCACCG 1114CAGCACCG 2051 AGCACCG 2988 hsa-miR-1251 ACUCUAGCUGCCAAAGGCGCU 178CAGCTAGAG 1115 AGCTAGAG 2052 GCTAGAG 2989 hsa-miR-1252AGAAGGAAAUUGAAUUCAUUUA 179 ATTTCCTTC 1116 TTTCCTTC 2053 TTCCTTC 2990hsa-miR-1253 AGAGAAGAAGAUCAGCCUGCA 180 CTTCTTCTC 1117 TTCTTCTC 2054TCTTCTC 2991 hsa-miR-1254 AGCCUGGAAGCUGGAGCCUGCAGU 181 CTTCCAGGC 1118TTCCAGGC 2055 TCCAGGC 2992 hsa-miR-1255a AGGAUGAGCAAAGAAAGUAGAUU 182TGCTCATCC 1119 GCTCATCC 2056 CTCATCC 2993 hsa-miR-1255bCGGAUGAGCAAAGAAAGUGGUU 183 TGCTCATCC 1120 GCTCATCC 2057 CTCATCC 2994hsa-miR-1256 AGGCAUUGACUUCUCACUAGCU 184 GTCAATGCC 1121 TCAATGCC 2058CAATGCC 2995 hsa-miR-1257 AGUGAAUGAUGGGUUCUGACC 185 ATCATTCAC 1122TCATTCAC 2059 CATTCAC 2996 hsa-miR-1258 AGUUAGGAUUAGGUCGUGGAA 186AATCCTAAC 1123 ATCCTAAC 2060 TCCTAAC 2997 hsa-miR-1259AUAUAUGAUGACUUAGCUUUU 187 CATCATATA 1124 ATCATATA 2061 TCATATA 2998hsa-miR-125a-3p ACAGGUGAGGUUCUUGGGAGCC 188 CCTCACCTG 1125 CTCACCTG 2062TCACCTG 2999 hsa-miR-125a-5p UCCCUGAGACCCUUUAACCUGUGA 189 GTCTCAGGG 1126TCTCAGGG 2063 CTCAGGG 3000 hsa-miR-125b UCCCUGAGACCCUAACUUGUGA 190GTCTCAGGG 1127 TCTCAGGG 2064 CTCAGGG 3001 hsa-miR-125b-1*ACGGGUUAGGCUCUUGGGAGCU 191 CCTAACCCG 1128 CTAACCCG 2065 TAACCCG 3002hsa-miR-125b-2* UCACAAGUCAGGCUCUUGGGAC 192 TGACTTGTG 1129 GACTTGTG 2066ACTTGTG 3003 hsa-miR-126 UCGUACCGUGAGUAAUAAUGCG 193 CACGGTACG 1130ACGGTACG 2067 CGGTACG 3004 hsa-miR-126* CAUUAUUACUUUUGGUACGCG 194AGTAATAAT 1131 GTAATAAT 2068 TAATAAT 3005 hsa-miR-1260AUCCCACCUCUGCCACCA 195 GAGGTGGGA 1132 AGGTGGGA 2069 GGTGGGA 3006hsa-miR-1261 AUGGAUAAGGCUUUGGCUU 196 CCTTATCCA 1133 CTTATCCA 2070TTATCCA 3007 hsa-miR-1262 AUGGGUGAAUUUGUAGAAGGAU 197 ATTCACCCA 1134TTCACCCA 2071 TCACCCA 3008 hsa-miR-1263 AUGGUACCCUGGCAUACUGAGU 198AGGGTACCA 1135 GGGTACCA 2072 GGTACCA 3009 hsa-miR-1264CAAGUCUUAUUUGAGCACCUGUU 199 ATAAGACTT 1136 TAAGACTT 2073 AAGACTT 3010hsa-miR-1265 CAGGAUGUGGUCAAGUGUUGUU 200 CCACATCCT 1137 CACATCCT 2074ACATCCT 3011 hsa-miR-1266 CCUCAGGGCUGUAGAACAGGGCU 201 AGCCCTGAG 1138GCCCTGAG 2075 CCCTGAG 3012 hsa-miR-1267 CCUGUUGAAGUGUAAUCCCCA 202CTTCAACAG 1139 TTCAACAG 2076 TCAACAG 3013 hsa-miR-1268CGGGCGUGGUGGUGGGGG 203 ACCACGCCC 1140 CCACGCCC 2077 CACGCCC 3014hsa-miR-1269 CUGGACUGAGCCGUGCUACUGG 204 CTCAGTCCA 1141 TCAGTCCA 2078CAGTCCA 3015 hsa-miR-127-3p UCGGAUCCGUCUGAGCUUGGCU 205 ACGGATCCG 1142CGGATCCG 2079 GGATCCG 3016 hsa-miR-127-5p CUGAAGCUCAGAGGGCUCUGAU 206TGAGCTTCA 1143 GAGCTTCA 2080 AGCTTCA 3017 hsa-miR-1270CUGGAGAUAUGGAAGAGCUGUGU 207 ATATCTCCA 1144 TATCTCCA 2081 ATCTCCA 3018hsa-miR-1271 CUUGGCACCUAGCAAGCACUCA 208 AGGTGCCAA 1145 GGTGCCAA 2082GTGCCAA 3019 hsa-miR-1272 GAUGAUGAUGGCAGCAAAUUCUGAAA 209 CATCATCAT 1146ATCATCAT 2083 TCATCAT 3020 hsa-miR-1273 GGGCGACAAAGCAAGACUCUUUCUU 210TTTGTCGCC 1147 TTGTCGCC 2084 TGTCGCC 3021 hsa-miR-1274aGUCCCUGUUCAGGCGCCA 211 GAACAGGGA 1148 AACAGGGA 2085 ACAGGGA 3022hsa-miR-1274b UCCCUGUUCGGGCGCCA 212 CGAACAGGG 1149 GAACAGGG 2086 AACAGGG3023 hsa-miR-1275 GUGGGGGAGAGGCUGUC 213 TCTCCCCCA 1150 CTCCCCCA 2087TCCCCCA 3024 hsa-miR-1276 UAAAGAGCCCUGUGGAGACA 214 GGGCTCTTT 1151GGCTCTTT 2088 GCTCTTT 3025 hsa-miR-1277 UACGUAGAUAUAUAUGUAUUUU 215TATCTACGT 1152 ATCTACGT 2089 TCTACGT 3026 hsa-miR-1278UAGUACUGUGCAUAUCAUCUAU 216 CACAGTACT 1153 ACAGTACT 2090 CAGTACT 3027hsa-miR-1279 UCAUAUUGCUUCUUUCU 217 AGCAATATG 1154 GCAATATG 2091 CAATATG3028 hsa-miR-128 UCACAGUGAACCGGUCUCUUU 218 TTCACTGTG 1155 TCACTGTG 2092CACTGTG 3029 hsa-miR-1280 UCCCACCGCUGCCACCC 219 AGCGGTGGG 1156 GCGGTGGG2093 CGGTGGG 3030 hsa-miR-1281 UCGCCUCCUCCUCUCCC 220 GAGGAGGCG 1157AGGAGGCG 2094 GGAGGCG 3031 hsa-miR-1282 UCGUUUGCCUUUUUCUGCUU 221AGGCAAACG 1158 GGCAAACG 2095 GCAAACG 3032 hsa-miR-1283UCUACAAAGGAAAGCGCUUUCU 222 CCTTTGTAG 1159 CTTTGTAG 2096 TTTGTAG 3033hsa-miR-1284 UCUAUACAGACCCUGGCUUUUC 223 TCTGTATAG 1160 CTGTATAG 2097TGTATAG 3034 hsa-miR-1285 UCUGGGCAACAAAGUGAGACCU 224 GTTGCCCAG 1161TTGCCCAG 2098 TGCCCAG 3035 hsa-miR-1286 UGCAGGACCAAGAUGAGCCCU 225TGGTCCTGC 1162 GGTCCTGC 2099 GTCCTGC 3036 hsa-miR-1287UGCUGGAUCAGUGGUUCGAGUC 226 TGATCCAGC 1163 GATCCAGC 2100 ATCCAGC 3037hsa-miR-1288 UGGACUGCCCUGAUCUGGAGA 227 GGGCAGTCC 1164 GGCAGTCC 2101GCAGTCC 3038 hsa-miR-1289 UGGAGUCCAGGAAUCUGCAUUUU 228 CTGGACTCC 1165TGGACTCC 2102 GGACTCC 3039 hsa-miR-129* AAGCCCUUACCCCAAAAAGUAU 229GTAAGGGCT 1166 TAAGGGCT 2103 AAGGGCT 3040 hsa-miR-129-3pAAGCCCUUACCCCAAAAAGCAU 230 GTAAGGGCT 1167 TAAGGGCT 2104 AAGGGCT 3041hsa-miR-129-5p CUUUUUGCGGUCUGGGCUUGC 231 CCGCAAAAA 1168 CGCAAAAA 2105GCAAAAA 3042 hsa-miR-1290 UGGAUUUUUGGAUCAGGGA 232 CAAAAATCC 1169AAAAATCC 2106 AAAATCC 3043 hsa-miR-1291 UGGCCCUGACUGAAGACCAGCAGU 233GTCAGGGCC 1170 TCAGGGCC 2107 CAGGGCC 3044 hsa-miR-1292UGGGAACGGGUUCCGGCAGACGCUG 234 CCCGTTCCC 1171 CCGTTCCC 2108 CGTTCCC 3045hsa-miR-1293 UGGGUGGUCUGGAGAUUUGUGC 235 AGACCACCC 1172 GACCACCC 2109ACCACCC 3046 hsa-miR-1294 UGUGAGGUUGGCAUUGUUGUCU 236 CAACCTCAC 1173AACCTCAC 2110 ACCTCAC 3047 hsa-miR-1295 UUAGGCCGCAGAUCUGGGUGA 237TGCGGCCTA 1174 GCGGCCTA 2111 CGGCCTA 3048 hsa-miR-1296UUAGGGCCCUGGCUCCAUCUCC 238 AGGGCCCTA 1175 GGGCCCTA 2112 GGCCCTA 3049hsa-miR-1297 UUCAAGUAAUUCAGGUG 239 ATTACTTGA 1176 TTACTTGA 2113 TACTTGA3050 hsa-miR-1298 UUCAUUCGGCUGUCCAGAUGUA 240 GCCGAATGA 1177 CCGAATGA2114 CGAATGA 3051 hsa-miR-1299 UUCUGGAAUUCUGUGUGAGGGA 241 AATTCCAGA 1178ATTCCAGA 2115 TTCCAGA 3052 hsa-miR-1300 UUGAGAAGGAGGCUGCUG 242 TCCTTCTCA1179 CCTTCTCA 2116 CTTCTCA 3053 hsa-miR-1301 UUGCAGCUGCCUGGGAGUGACUUC243 GCAGCTGCA 1180 CAGCTGCA 2117 AGCTGCA 3054 hsa-miR-1302UUGGGACAUACUUAUGCUAAA 244 TATGTCCCA 1181 ATGTCCCA 2118 TGTCCCA 3055hsa-miR-1303 UUUAGAGACGGGGUCUUGCUCU 245 CGTCTCTAA 1182 GTCTCTAA 2119TCTCTAA 3056 hsa-miR-1304 UUUGAGGCUACAGUGAGAUGUG 246 TAGCCTCAA 1183AGCCTCAA 2120 GCCTCAA 3057 hsa-miR-1305 UUUUCAACUCUAAUGGGAGAGA 247GAGTTGAAA 1184 AGTTGAAA 2121 GTTGAAA 3058 hsa-miR-1306ACGUUGGCUCUGGUGGUG 248 GAGCCAACG 1185 AGCCAACG 2122 GCCAACG 3059hsa-miR-1307 ACUCGGCGUGGCGUCGGUCGUG 249 CACGCCGAG 1186 ACGCCGAG 2123CGCCGAG 3060 hsa-miR-1308 GCAUGGGUGGUUCAGUGG 250 CCACCCATG 1187 CACCCATG2124 ACCCATG 3061 hsa-miR-130a CAGUGCAAUGUUAAAAGGGCAU 251 CATTGCACT 1188ATTGCACT 2125 TTGCACT 3062 hsa-miR-130a* UUCACAUUGUGCUACUGUCUGC 252ACAATGTGA 1189 CAATGTGA 2126 AATGTGA 3063 hsa-miR-130bCAGUGCAAUGAUGAAAGGGCAU 253 CATTGCACT 1190 ATTGCACT 2127 TTGCACT 3064hsa-miR-130b* ACUCUUUCCCUGUUGCACUAC 254 GGGAAAGAG 1191 GGAAAGAG 2128GAAAGAG 3065 hsa-miR-132 UAACAGUCUACAGCCAUGGUCG 255 TAGACTGTT 1192AGACTGTT 2129 GACTGTT 3066 hsa-miR-132* ACCGUGGCUUUCGAUUGUUACU 256AAGCCACGG 1193 AGCCACGG 2130 GCCACGG 3067 hsa-miR-1321CAGGGAGGUGAAUGUGAU 257 CACCTCCCT 1194 ACCTCCCT 2131 CCTCCCT 3068hsa-miR-1322 GAUGAUGCUGCUGAUGCUG 258 CAGCATCAT 1195 AGCATCAT 2132GCATCAT 3069 hsa-miR-1323 UCAAAACUGAGGGGCAUUUUCU 259 TCAGTTTTG 1196CAGTTTTG 2133 AGTTTTG 3070 hsa-miR-1324 CCAGACAGAAUUCUAUGCACUUUC 260TTCTGTCTG 1197 TCTGTCTG 2134 CTGTCTG 3071 hsa-miR-133aUUUGGUCCCCUUCAACCAGCUG 261 GGGGACCAA 1198 GGGACCAA 2135 GGACCAA 3072hsa-miR-133b UUUGGUCCCCUUCAACCAGCUA 262 GGGGACCAA 1199 GGGACCAA 2136GGACCAA 3073 hsa-miR-134 UGUGACUGGUUGACCAGAGGGG 263 ACCAGTCAC 1200CCAGTCAC 2137 CAGTCAC 3074 hsa-miR-135a UAUGGCUUUUUAUUCCUAUGUGA 264AAAAGCCAT 1201 AAAGCCAT 2138 AAGCCAT 3075 hsa-miR-135a*UAUAGGGAUUGGAGCCGUGGCG 265 AATCCCTAT 1202 ATCCCTAT 2139 TCCCTAT 3076hsa-miR-135b UAUGGCUUUUCAUUCCUAUGUGA 266 AAAAGCCAT 1203 AAAGCCAT 2140AAGCCAT 3077 hsa-miR-135b* AUGUAGGGCUAAAAGCCAUGGG 267 AGCCCTACA 1204GCCCTACA 2141 CCCTACA 3078 hsa-miR-136 ACUCCAUUUGUUUUGAUGAUGGA 268CAAATGGAG 1205 AAATGGAG 2142 AATGGAG 3079 hsa-miR-136*CAUCAUCGUCUCAAAUGAGUCU 269 GACGATGAT 1206 ACGATGAT 2143 CGATGAT 3080hsa-miR-137 UUAUUGCUUAAGAAUACGCGUAG 270 TAAGCAATA 1207 AAGCAATA 2144AGCAATA 3081 hsa-miR-138 AGCUGGUGUUGUGAAUCAGGCCG 271 AACACCAGC 1208ACACCAGC 2145 CACCAGC 3082 hsa-miR-138-1* GCUACUUCACAACACCAGGGCC 272GTGAAGTAG 1209 TGAAGTAG 2146 GAAGTAG 3083 hsa-miR-138-2*GCUAUUUCACGACACCAGGGUU 273 GTGAAATAG 1210 TGAAATAG 2147 GAAATAG 3084hsa-miR-139-3p GGAGACGCGGCCCUGUUGGAGU 274 CCGCGTCTC 1211 CGCGTCTC 2148GCGTCTC 3085 hsa-miR-139-5p UCUACAGUGCACGUGUCUCCAG 275 GCACTGTAG 1212CACTGTAG 2149 ACTGTAG 3086 hsa-miR-140-3p UACCACAGGGUAGAACCACGG 276CCCTGTGGT 1213 CCTGTGGT 2150 CTGTGGT 3087 hsa-miR-140-5pCAGUGGUUUUACCCUAUGGUAG 277 AAAACCACT 1214 AAACCACT 2151 AACCACT 3088hsa-miR-141 UAACACUGUCUGGUAAAGAUGG 278 GACAGTGTT 1215 ACAGTGTT 2152CAGTGTT 3089 hsa-miR-141* CAUCUUCCAGUACAGUGUUGGA 279 CTGGAAGAT 1216TGGAAGAT 2153 GGAAGAT 3090 hsa-miR-142-3p UGUAGUGUUUCCUACUUUAUGGA 280AAACACTAC 1217 AACACTAC 2154 ACACTAC 3091 hsa-miR-142-5pCAUAAAGUAGAAAGCACUACU 281 CTACTTTAT 1218 TACTTTAT 2155 ACTTTAT 3092hsa-miR-143 UGAGAUGAAGCACUGUAGCUC 282 CTTCATCTC 1219 TTCATCTC 2156TCATCTC 3093 hsa-miR-143* GGUGCAGUGCUGCAUCUCUGGU 283 GCACTGCAC 1220CACTGCAC 2157 ACTGCAC 3094 hsa-miR-144 UACAGUAUAGAUGAUGUACU 284CTATACTGT 1221 TATACTGT 2158 ATACTGT 3095 hsa-miR-144*GGAUAUCAUCAUAUACUGUAAG 285 GATGATATC 1222 ATGATATC 2159 TGATATC 3096hsa-miR-145 GUCCAGUUUUCCCAGGAAUCCCU 286 AAAACTGGA 1223 AAACTGGA 2160AACTGGA 3097 hsa-miR-145* GGAUUCCUGGAAAUACUGUUCU 287 CCAGGAATC 1224CAGGAATC 2161 AGGAATC 3098 hsa-miR-1468 CUCCGUUUGCCUGUUUCGCUG 288GCAAACGGA 1225 CAAACGGA 2162 AAACGGA 3099 hsa-miR-1469CUCGGCGCGGGGCGCGGGCUCC 289 CCGCGCCGA 1226 CGCGCCGA 2163 GCGCCGA 3100hsa-miR-146a UGAGAACUGAAUUCCAUGGGUU 290 TCAGTTCTC 1227 CAGTTCTC 2164AGTTCTC 3101 hsa-miR-146a* CCUCUGAAAUUCAGUUCUUCAG 291 ATTTCAGAG 1228TTTCAGAG 2165 TTCAGAG 3102 hsa-miR-146b-3p UGCCCUGUGGACUCAGUUCUGG 292CCACAGGGC 1229 CACAGGGC 2166 ACAGGGC 3103 hsa-miR-146b-5pUGAGAACUGAAUUCCAUAGGCU 293 TCAGTTCTC 1230 CAGTTCTC 2167 AGTTCTC 3104hsa-miR-147 GUGUGUGGAAAUGCUUCUGC 294 TTCCACACA 1231 TCCACACA 2168CCACACA 3105 hsa-miR-1470 GCCCUCCGCCCGUGCACCCCG 295 GGCGGAGGG 1232GCGGAGGG 2169 CGGAGGG 3106 hsa-miR-1471 GCCCGCGUGUGGAGCCAGGUGU 296ACACGCGGG 1233 CACGCGGG 2170 ACGCGGG 3107 hsa-miR-147bGUGUGCGGAAAUGCUUCUGCUA 297 TTCCGCACA 1234 TCCGCACA 2171 CCGCACA 3108hsa-miR-148a UCAGUGCACUACAGAACUUUGU 298 AGTGCACTG 1235 GTGCACTG 2172TGCACTG 3109 hsa-miR-148a* AAAGUUCUGAGACACUCCGACU 299 TCAGAACTT 1236CAGAACTT 2173 AGAACTT 3110 hsa-miR-148b UCAGUGCAUCACAGAACUUUGU 300GATGCACTG 1237 ATGCACTG 2174 TGCACTG 3111 hsa-miR-148b*AAGUUCUGUUAUACACUCAGGC 301 AACAGAACT 1238 ACAGAACT 2175 CAGAACT 3112hsa-miR-149 UCUGGCUCCGUGUCUUCACUCCC 302 CGGAGCCAG 1239 GGAGCCAG 2176GAGCCAG 3113 hsa-miR-149* AGGGAGGGACGGGGGCUGUGC 303 GTCCCTCCC 1240TCCCTCCC 2177 CCCTCCC 3114 hsa-miR-150 UCUCCCAACCCUUGUACCAGUG 304GGTTGGGAG 1241 GTTGGGAG 2178 TTGGGAG 3115 hsa-miR-150*CUGGUACAGGCCUGGGGGACAG 305 CCTGTACCA 1242 CTGTACCA 2179 TGTACCA 3116hsa-miR-151-3p CUAGACUGAAGCUCCUUGAGG 306 TTCAGTCTA 1243 TCAGTCTA 2180CAGTCTA 3117 hsa-miR-151-5p UCGAGGAGCUCACAGUCUAGU 307 AGCTCCTCG 1244GCTCCTCG 2181 CTCCTCG 3118 hsa-miR-152 UCAGUGCAUGACAGAACUUGG 308CATGCACTG 1245 ATGCACTG 2182 TGCACTG 3119 hsa-miR-153UUGCAUAGUCACAAAAGUGAUC 309 GACTATGCA 1246 ACTATGCA 2183 CTATGCA 3120hsa-miR-1537 AAAACCGUCUAGUUACAGUUGU 310 AGACGGTTT 1247 GACGGTTT 2184ACGGTTT 3121 hsa-miR-1538 CGGCCCGGGCUGCUGCUGUUCCU 311 GCCCGGGCC 1248CCCGGGCC 2185 CCGGGCC 3122 hsa-miR-1539 UCCUGCGCGUCCCAGAUGCCC 312ACGCGCAGG 1249 CGCGCAGG 2186 GCGCAGG 3123 hsa-miR-154UAGGUUAUCCGUGUUGCCUUCG 313 GGATAACCT 1250 GATAACCT 2187 ATAACCT 3124hsa-miR-154* AAUCAUACACGGUUGACCUAUU 314 GTGTATGAT 1251 TGTATGAT 2188GTATGAT 3125 hsa-miR-155 UUAAUGCUAAUCGUGAUAGGGGU 315 TTAGCATTA 1252TAGCATTA 2189 AGCATTA 3126 hsa-miR-155* CUCCUACAUAUUAGCAUUAACA 316TATGTAGGA 1253 ATGTAGGA 2190 TGTAGGA 3127 hsa-miR-15aUAGCAGCACAUAAUGGUUUGUG 317 TGTGCTGCT 1254 GTGCTGCT 2191 TGCTGCT 3128hsa-miR-15a* CAGGCCAUAUUGUGCUGCCUCA 318 ATATGGCCT 1255 TATGGCCT 2192ATGGCCT 3129 hsa-miR-15b UAGCAGCACAUCAUGGUUUACA 319 TGTGCTGCT 1256GTGCTGCT 2193 TGCTGCT 3130 hsa-miR-15b* CGAAUCAUUAUUUGCUGCUCUA 320TAATGATTC 1257 AATGATTC 2194 ATGATTC 3131 hsa-miR-16UAGCAGCACGUAAAUAUUGGCG 321 CGTGCTGCT 1258 GTGCTGCT 2195 TGCTGCT 3132hsa-miR-16-1* CCAGUAUUAACUGUGCUGCUGA 322 TTAATACTG 1259 TAATACTG 2196AATACTG 3133 hsa-miR-16-2* CCAAUAUUACUGUGCUGCUUUA 323 GTAATATTG 1260TAATATTG 2197 AATATTG 3134 hsa-miR-17 CAAAGUGCUUACAGUGCAGGUAG 324AAGCACTTT 1261 AGCACTTT 2198 GCACTTT 3135 hsa-miR-17*ACUGCAGUGAAGGCACUUGUAG 325 TCACTGCAG 1262 CACTGCAG 2199 ACTGCAG 3136hsa-miR-181a AACAUUCAACGCUGUCGGUGAGU 326 GTTGAATGT 1263 TTGAATGT 2200TGAATGT 3137 hsa-miR-181a* ACCAUCGACCGUUGAUUGUACC 327 GGTCGATGG 1264GTCGATGG 2201 TCGATGG 3138 hsa-miR-181a-2* ACCACUGACCGUUGACUGUACC 328GGTCAGTGG 1265 GTCAGTGG 2202 TCAGTGG 3139 hsa-miR-181bAACAUUCAUUGCUGUCGGUGGGU 329 AATGAATGT 1266 ATGAATGT 2203 TGAATGT 3140hsa-miR-181c AACAUUCAACCUGUCGGUGAGU 330 GTTGAATGT 1267 TTGAATGT 2204TGAATGT 3141 hsa-miR-181c* AACCAUCGACCGUUGAGUGGAC 331 GTCGATGGT 1268TCGATGGT 2205 CGATGGT 3142 hsa-miR-181d AACAUUCAUUGUUGUCGGUGGGU 332AATGAATGT 1269 ATGAATGT 2206 TGAATGT 3143 hsa-miR-182UUUGGCAAUGGUAGAACUCACACU 333 CATTGCCAA 1270 ATTGCCAA 2207 TTGCCAA 3144hsa-miR-182* UGGUUCUAGACUUGCCAACUA 334 TCTAGAACC 1271 CTAGAACC 2208TAGAACC 3145 hsa-miR-1825 UCCAGUGCCCUCCUCUCC 335 GGGCACTGG 1272 GGCACTGG2209 GCACTGG 3146 hsa-miR-1826 AUUGAUCAUCGACACUUCGAACGCAAU 336 GATGATCAA1273 ATGATCAA 2210 TGATCAA 3147 hsa-miR-1827 UGAGGCAGUAGAUUGAAU 337TACTGCCTC 1274 ACTGCCTC 2211 CTGCCTC 3148 hsa-miR-183UAUGGCACUGGUAGAAUUCACU 338 CAGTGCCAT 1275 AGTGCCAT 2212 GTGCCAT 3149hsa-miR-183* GUGAAUUACCGAAGGGCCAUAA 339 GGTAATTCA 1276 GTAATTCA 2213TAATTCA 3150 hsa-miR-184 UGGACGGAGAACUGAUAAGGGU 340 TCTCCGTCC 1277CTCCGTCC 2214 TCCGTCC 3151 hsa-miR-185 UGGAGAGAAAGGCAGUUCCUGA 341TTTCTCTCC 1278 TTCTCTCC 2215 TCTCTCC 3152 hsa-miR-185*AGGGGCUGGCUUUCCUCUGGUC 342 GCCAGCCCC 1279 CCAGCCCC 2216 CAGCCCC 3153hsa-miR-186 CAAAGAAUUCUCCUUUUGGGCU 343 GAATTCTTT 1280 AATTCTTT 2217ATTCTTT 3154 hsa-miR-186* GCCCAAAGGUGAAUUUUUUGGG 344 ACCTTTGGG 1281CCTTTGGG 2218 CTTTGGG 3155 hsa-miR-187 UCGUGUCUUGUGUUGCAGCCGG 345CAAGACACG 1282 AAGACACG 2219 AGACACG 3156 hsa-miR-187*GGCUACAACACAGGACCCGGGC 346 TGTTGTAGC 1283 GTTGTAGC 2220 TTGTAGC 3157hsa-miR-188-3p CUCCCACAUGCAGGGUUUGCA 347 CATGTGGGA 1284 ATGTGGGA 2221TGTGGGA 3158 hsa-miR-188-5p CAUCCCUUGCAUGGUGGAGGG 348 GCAAGGGAT 1285CAAGGGAT 2222 AAGGGAT 3159 hsa-miR-18a UAAGGUGCAUCUAGUGCAGAUAG 349ATGCACCTT 1286 TGCACCTT 2223 GCACCTT 3160 hsa-miR-18a*ACUGCCCUAAGUGCUCCUUCUGG 350 TTAGGGCAG 1287 TAGGGCAG 2224 AGGGCAG 3161hsa-miR-18b UAAGGUGCAUCUAGUGCAGUUAG 351 ATGCACCTT 1288 TGCACCTT 2225GCACCTT 3162 hsa-miR-18b* UGCCCUAAAUGCCCCUUCUGGC 352 ATTTAGGGC 1289TTTAGGGC 2226 TTAGGGC 3163 hsa-miR-190 UGAUAUGUUUGAUAUAUUAGGU 353AAACATATC 1290 AACATATC 2227 ACATATC 3164 hsa-miR-1908CGGCGGGGACGGCGAUUGGUC 354 GTCCCCGCC 1291 TCCCCGCC 2228 CCCCGCC 3165hsa-miR-1909 CGCAGGGGCCGGGUGCUCACCG 355 GGCCCCTGC 1292 GCCCCTGC 2229CCCCTGC 3166 hsa-miR-1909* UGAGUGCCGGUGCCUGCCCUG 356 CCGGCACTC 1293CGGCACTC 2230 GGCACTC 3167 hsa-miR-190b UGAUAUGUUUGAUAUUGGGUU 357AAACATATC 1294 AACATATC 2231 ACATATC 3168 hsa-miR-191CAACGGAAUCCCAAAAGCAGCUG 358 GATTCCGTT 1295 ATTCCGTT 2232 TTCCGTT 3169hsa-miR-191* GCUGCGCUUGGAUUUCGUCCCC 359 CAAGCGCAG 1296 AAGCGCAG 2233AGCGCAG 3170 hsa-miR-1910 CCAGUCCUGUGCCUGCCGCCU 360 ACAGGACTG 1297CAGGACTG 2234 AGGACTG 3171 hsa-miR-1911 UGAGUACCGCCAUGUCUGUUGGG 361GCGGTACTC 1298 CGGTACTC 2235 GGTACTC 3172 hsa-miR-1911*CACCAGGCAUUGUGGUCUCC 362 ATGCCTGGT 1299 TGCCTGGT 2236 GCCTGGT 3173hsa-miR-1912 UACCCAGAGCAUGCAGUGUGAA 363 GCTCTGGGT 1300 CTCTGGGT 2237TCTGGGT 3174 hsa-miR-1913 UCUGCCCCCUCCGCUGCUGCCA 364 AGGGGGCAG 1301GGGGGCAG 2238 GGGGCAG 3175 hsa-miR-1914 CCCUGUGCCCGGCCCACUUCUG 365GGGCACAGG 1302 GGCACAGG 2239 GCACAGG 3176 hsa-miR-1914*GGAGGGGUCCCGCACUGGGAGG 366 GGACCCCTC 1303 GACCCCTC 2240 ACCCCTC 3177hsa-miR-1915 CCCCAGGGCGACGCGGCGGG 367 CGCCCTGGG 1304 GCCCTGGG 2241CCCTGGG 3178 hsa-miR-1915* ACCUUGCCUUGCUGCCCGGGCC 368 AAGGCAAGG 1305AGGCAAGG 2242 GGCAAGG 3179 hsa-miR-192 CUGACCUAUGAAUUGACAGCC 369CATAGGTCA 1306 ATAGGTCA 2243 TAGGTCA 3180 hsa-miR-192*CUGCCAAUUCCAUAGGUCACAG 370 GAATTGGCA 1307 AATTGGCA 2244 ATTGGCA 3181hsa-miR-193a-3p AACUGGCCUACAAAGUCCCAGU 371 TAGGCCAGT 1308 AGGCCAGT 2245GGCCAGT 3182 hsa-miR-193a-5p UGGGUCUUUGCGGGCGAGAUGA 372 CAAAGACCC 1309AAAGACCC 2246 AAGACCC 3183 hsa-miR-193b AACUGGCCCUCAAAGUCCCGCU 373AGGGCCAGT 1310 GGGCCAGT 2247 GGCCAGT 3184 hsa-miR-193b*CGGGGUUUUGAGGGCGAGAUGA 374 CAAAACCCC 1311 AAAACCCC 2248 AAACCCC 3185hsa-miR-194 UGUAACAGCAACUCCAUGUGGA 375 TGCTGTTAC 1312 GCTGTTAC 2249CTGTTAC 3186 hsa-miR-194* CCAGUGGGGCUGCUGUUAUCUG 376 GCCCCACTG 1313CCCCACTG 2250 CCCACTG 3187 hsa-miR-195 UAGCAGCACAGAAAUAUUGGC 377TGTGCTGCT 1314 GTGCTGCT 2251 TGCTGCT 3188 hsa-miR-195*CCAAUAUUGGCUGUGCUGCUCC 378 CCAATATTG 1315 CAATATTG 2252 AATATTG 3189hsa-miR-196a UAGGUAGUUUCAUGUUGUUGGG 379 AAACTACCT 1316 AACTACCT 2253ACTACCT 3190 hsa-miR-196a* CGGCAACAAGAAACUGCCUGAG 380 CTTGTTGCC 1317TTGTTGCC 2254 TGTTGCC 3191 hsa-miR-196b UAGGUAGUUUCCUGUUGUUGGG 381AAACTACCT 1318 AACTACCT 2255 ACTACCT 3192 hsa-miR-197UUCACCACCUUCUCCACCCAGC 382 AGGTGGTGA 1319 GGTGGTGA 2256 GTGGTGA 3193hsa-miR-198 GGUCCAGAGGGGAGAUAGGUUC 383 CCTCTGGAC 1320 CTCTGGAC 2257TCTGGAC 3194 hsa-miR-199a-5p CCCAGUGUUCAGACUACCUGUUC 384 GAACACTGG 1321AACACTGG 2258 ACACTGG 3195 hsa-miR-199b-3p ACAGUAGUCUGCACAUUGGUUA 385AGACTACTG 1322 GACTACTG 2259 ACTACTG 3196 hsa-miR-199b-5pCCCAGUGUUUAGACUAUCUGUUC 386 AAACACTGG 1323 AACACTGG 2260 ACACTGG 3197hsa-miR-19a UGUGCAAAUCUAUGCAAAACUGA 387 GATTTGCAC 1324 ATTTGCAC 2261TTTGCAC 3198 hsa-miR-19a* AGUUUUGCAUAGUUGCACUACA 388 ATGCAAAAC 1325TGCAAAAC 2262 GCAAAAC 3199 hsa-miR-19b UGUGCAAAUCCAUGCAAAACUGA 389GATTTGCAC 1326 ATTTGCAC 2263 TTTGCAC 3200 hsa-miR-19b-1*AGUUUUGCAGGUUUGCAUCCAGC 390 CTGCAAAAC 1327 TGCAAAAC 2264 GCAAAAC 3201hsa-miR-19b-2* AGUUUUGCAGGUUUGCAUUUCA 391 CTGCAAAAC 1328 TGCAAAAC 2265GCAAAAC 3202 hsa-miR-200a UAACACUGUCUGGUAACGAUGU 392 GACAGTGTT 1329ACAGTGTT 2266 CAGTGTT 3203 hsa-miR-200a* CAUCUUACCGGACAGUGCUGGA 393CGGTAAGAT 1330 GGTAAGAT 2267 GTAAGAT 3204 hsa-miR-200bUAAUACUGCCUGGUAAUGAUGA 394 GGCAGTATT 1331 GCAGTATT 2268 CAGTATT 3205hsa-miR-200b* CAUCUUACUGGGCAGCAUUGGA 395 CAGTAAGAT 1332 AGTAAGAT 2269GTAAGAT 3206 hsa-miR-200c UAAUACUGCCGGGUAAUGAUGGA 396 GGCAGTATT 1333GCAGTATT 2270 CAGTATT 3207 hsa-miR-200c* CGUCUUACCCAGCAGUGUUUGG 397GGGTAAGAC 1334 GGTAAGAC 2271 GTAAGAC 3208 hsa-miR-202AGAGGUAUAGGGCAUGGGAA 398 CTATACCTC 1335 TATACCTC 2272 ATACCTC 3209hsa-miR-202* UUCCUAUGCAUAUACUUCUUUG 399 TGCATAGGA 1336 GCATAGGA 2273CATAGGA 3210 hsa-miR-203 GUGAAAUGUUUAGGACCACUAG 400 AACATTTCA 1337ACATTTCA 2274 CATTTCA 3211 hsa-miR-204 UUCCCUUUGUCAUCCUAUGCCU 401ACAAAGGGA 1338 CAAAGGGA 2275 AAAGGGA 3212 hsa-miR-205UCCUUCAUUCCACCGGAGUCUG 402 GAATGAAGG 1339 AATGAAGG 2276 ATGAAGG 3213hsa-miR-206 UGGAAUGUAAGGAAGUGUGUGG 403 TTACATTCC 1340 TACATTCC 2277ACATTCC 3214 hsa-miR-208a AUAAGACGAGCAAAAAGCUUGU 404 CTCGTCTTA 1341TCGTCTTA 2278 CGTCTTA 3215 hsa-miR-208b AUAAGACGAACAAAAGGUUUGU 405TTCGTCTTA 1342 TCGTCTTA 2279 CGTCTTA 3216 hsa-miR-20aUAAAGUGCUUAUAGUGCAGGUAG 406 AAGCACTTT 1343 AGCACTTT 2280 GCACTTT 3217hsa-miR-20a* ACUGCAUUAUGAGCACUUAAAG 407 ATAATGCAG 1344 TAATGCAG 2281AATGCAG 3218 hsa-miR-20b CAAAGUGCUCAUAGUGCAGGUAG 408 GAGCACTTT 1345AGCACTTT 2282 GCACTTT 3219 hsa-miR-20b* ACUGUAGUAUGGGCACUUCCAG 409ATACTACAG 1346 TACTACAG 2283 ACTACAG 3220 hsa-miR-21UAGCUUAUCAGACUGAUGUUGA 410 TGATAAGCT 1347 GATAAGCT 2284 ATAAGCT 3221hsa-miR-21* CAACACCAGUCGAUGGGCUGU 411 ACTGGTGTT 1348 CTGGTGTT 2285TGGTGTT 3222 hsa-miR-210 CUGUGCGUGUGACAGCGGCUGA 412 ACACGCACA 1349CACGCACA 2286 ACGCACA 3223 hsa-miR-211 UUCCCUUUGUCAUCCUUCGCCU 413ACAAAGGGA 1350 CAAAGGGA 2287 AAAGGGA 3224 hsa-miR-212UAACAGUCUCCAGUCACGGCC 414 GAGACTGTT 1351 AGACTGTT 2288 GACTGTT 3225hsa-miR-214 ACAGCAGGCACAGACAGGCAGU 415 TGCCTGCTG 1352 GCCTGCTG 2289CCTGCTG 3226 hsa-miR-214* UGCCUGUCUACACUUGCUGUGC 416 TAGACAGGC 1353AGACAGGC 2290 GACAGGC 3227 hsa-miR-215 AUGACCUAUGAAUUGACAGAC 417CATAGGTCA 1354 ATAGGTCA 2291 TAGGTCA 3228 hsa-miR-216aUAAUCUCAGCUGGCAACUGUGA 418 GCTGAGATT 1355 CTGAGATT 2292 TGAGATT 3229hsa-miR-216b AAAUCUCUGCAGGCAAAUGUGA 419 GCAGAGATT 1356 CAGAGATT 2293AGAGATT 3230 hsa-miR-217 UACUGCAUCAGGAACUGAUUGGA 420 TGATGCAGT 1357GATGCAGT 2294 ATGCAGT 3231 hsa-miR-218 UUGUGCUUGAUCUAACCAUGU 421TCAAGCACA 1358 CAAGCACA 2295 AAGCACA 3232 hsa-miR-218-1*AUGGUUCCGUCAAGCACCAUGG 422 ACGGAACCA 1359 CGGAACCA 2296 GGAACCA 3233hsa-miR-218-2* CAUGGUUCUGUCAAGCACCGCG 423 CAGAACCAT 1360 AGAACCAT 2297GAACCAT 3234 hsa-miR-219-1-3p AGAGUUGAGUCUGGACGUCCCG 424 ACTCAACTC 1361CTCAACTC 2298 TCAACTC 3235 hsa-miR-219-2-3p AGAAUUGUGGCUGGACAUCUGU 425CCACAATTC 1362 CACAATTC 2299 ACAATTC 3236 hsa-miR-219-5pUGAUUGUCCAAACGCAAUUCU 426 TGGACAATC 1363 GGACAATC 2300 GACAATC 3237hsa-miR-22 AAGCUGCCAGUUGAAGAACUGU 427 CTGGCAGCT 1364 TGGCAGCT 2301GGCAGCT 3238 hsa-miR-22* AGUUCUUCAGUGGCAAGCUUUA 428 CTGAAGAAC 1365TGAAGAAC 2302 GAAGAAC 3239 hsa-miR-220a CCACACCGUAUCUGACACUUU 429TACGGTGTG 1366 ACGGTGTG 2303 CGGTGTG 3240 hsa-miR-220bCCACCACCGUGUCUGACACUU 430 ACGGTGGTG 1367 CGGTGGTG 2304 GGTGGTG 3241hsa-miR-220c ACACAGGGCUGUUGUGAAGACU 431 AGCCCTGTG 1368 GCCCTGTG 2305CCCTGTG 3242 hsa-miR-221 AGCUACAUUGUCUGCUGGGUUUC 432 CAATGTAGC 1369AATGTAGC 2306 ATGTAGC 3243 hsa-miR-221* ACCUGGCAUACAAUGUAGAUUU 433TATGCCAGG 1370 ATGCCAGG 2307 TGCCAGG 3244 hsa-miR-222AGCUACAUCUGGCUACUGGGU 434 AGATGTAGC 1371 GATGTAGC 2308 ATGTAGC 3245hsa-miR-222* CUCAGUAGCCAGUGUAGAUCCU 435 GGCTACTGA 1372 GCTACTGA 2309CTACTGA 3246 hsa-miR-223 UGUCAGUUUGUCAAAUACCCCA 436 CAAACTGAC 1373AAACTGAC 2310 AACTGAC 3247 hsa-miR-223* CGUGUAUUUGACAAGCUGAGUU 437CAAATACAC 1374 AAATACAC 2311 AATACAC 3248 hsa-miR-224CAAGUCACUAGUGGUUCCGUU 438 TAGTGACTT 1375 AGTGACTT 2312 GTGACTT 3249hsa-miR-23a AUCACAUUGCCAGGGAUUUCC 439 GCAATGTGA 1376 CAATGTGA 2313AATGTGA 3250 hsa-miR-23a* GGGGUUCCUGGGGAUGGGAUUU 440 CAGGAACCC 1377AGGAACCC 2314 GGAACCC 3251 hsa-miR-23b AUCACAUUGCCAGGGAUUACC 441GCAATGTGA 1378 CAATGTGA 2315 AATGTGA 3252 hsa-miR-23b*UGGGUUCCUGGCAUGCUGAUUU 442 CAGGAACCC 1379 AGGAACCC 2316 GGAACCC 3253hsa-miR-24 UGGCUCAGUUCAGCAGGAACAG 443 AACTGAGCC 1380 ACTGAGCC 2317CTGAGCC 3254 hsa-miR-24-1* UGCCUACUGAGCUGAUAUCAGU 444 TCAGTAGGC 1381CAGTAGGC 2318 AGTAGGC 3255 hsa-miR-24-2* UGCCUACUGAGCUGAAACACAG 445TCAGTAGGC 1382 CAGTAGGC 2319 AGTAGGC 3256 hsa-miR-25CAUUGCACUUGUCUCGGUCUGA 446 AAGTGCAAT 1383 AGTGCAAT 2320 GTGCAAT 3257hsa-miR-25* AGGCGGAGACUUGGGCAAUUG 447 GTCTCCGCC 1384 TCTCCGCC 2321CTCCGCC 3258 hsa-miR-26a UUCAAGUAAUCCAGGAUAGGCU 448 ATTACTTGA 1385TTACTTGA 2322 TACTTGA 3259 hsa-miR-26a-1* CCUAUUCUUGGUUACUUGCACG 449CAAGAATAG 1386 AAGAATAG 2323 AGAATAG 3260 hsa-miR-26a-2*CCUAUUCUUGAUUACUUGUUUC 450 CAAGAATAG 1387 AAGAATAG 2324 AGAATAG 3261hsa-miR-26b UUCAAGUAAUUCAGGAUAGGU 451 ATTACTTGA 1388 TTACTTGA 2325TACTTGA 3262 hsa-miR-26b* CCUGUUCUCCAUUACUUGGCUC 452 GGAGAACAG 1389GAGAACAG 2326 AGAACAG 3263 hsa-miR-27a UUCACAGUGGCUAAGUUCCGC 453CCACTGTGA 1390 CACTGTGA 2327 ACTGTGA 3264 hsa-miR-27a*AGGGCUUAGCUGCUUGUGAGCA 454 GCTAAGCCC 1391 CTAAGCCC 2328 TAAGCCC 3265hsa-miR-27b UUCACAGUGGCUAAGUUCUGC 455 CCACTGTGA 1392 CACTGTGA 2329ACTGTGA 3266 hsa-miR-27b* AGAGCUUAGCUGAUUGGUGAAC 456 GCTAAGCTC 1393CTAAGCTC 2330 TAAGCTC 3267 hsa-miR-28-3p CACUAGAUUGUGAGCUCCUGGA 457CAATCTAGT 1394 AATCTAGT 2331 ATCTAGT 3268 hsa-miR-28-5pAAGGAGCUCACAGUCUAUUGAG 458 TGAGCTCCT 1395 GAGCTCCT 2332 AGCTCCT 3269hsa-miR-296-3p GAGGGUUGGGUGGAGGCUCUCC 459 CCCAACCCT 1396 CCAACCCT 2333CAACCCT 3270 hsa-miR-296-5p AGGGCCCCCCCUCAAUCCUGU 460 GGGGGGCCC 1397GGGGGCCC 2334 GGGGCCC 3271 hsa-miR-297 AUGUAUGUGUGCAUGUGCAUG 461ACACATACA 1398 CACATACA 2335 ACATACA 3272 hsa-miR-298AGCAGAAGCAGGGAGGUUCUCCCA 462 TGCTTCTGC 1399 GCTTCTGC 2336 CTTCTGC 3273hsa-miR-299-3p UAUGUGGGAUGGUAAACCGCUU 463 ATCCCACAT 1400 TCCCACAT 2337CCCACAT 3274 hsa-miR-299-5p UGGUUUACCGUCCCACAUACAU 464 CGGTAAACC 1401GGTAAACC 2338 GTAAACC 3275 hsa-miR-29a UAGCACCAUCUGAAAUCGGUUA 465GATGGTGCT 1402 ATGGTGCT 2339 TGGTGCT 3276 hsa-miR-29a*ACUGAUUUCUUUUGGUGUUCAG 466 AGAAATCAG 1403 GAAATCAG 2340 AAATCAG 3277hsa-miR-29b UAGCACCAUUUGAAAUCAGUGUU 467 AATGGTGCT 1404 ATGGTGCT 2341TGGTGCT 3278 hsa-miR-29b-1* GCUGGUUUCAUAUGGUGGUUUAGA 468 TGAAACCAG 1405GAAACCAG 2342 AAACCAG 3279 hsa-miR-29b-2* CUGGUUUCACAUGGUGGCUUAG 469GTGAAACCA 1406 TGAAACCA 2343 GAAACCA 3280 hsa-miR-29cUAGCACCAUUUGAAAUCGGUUA 470 AATGGTGCT 1407 ATGGTGCT 2344 TGGTGCT 3281hsa-miR-29c* UGACCGAUUUCUCCUGGUGUUC 471 AAATCGGTC 1408 AATCGGTC 2345ATCGGTC 3282 hsa-miR-300 UAUACAAGGGCAGACUCUCUCU 472 CCCTTGTAT 1409CCTTGTAT 2346 CTTGTAT 3283 hsa-miR-301a CAGUGCAAUAGUAUUGUCAAAGC 473TATTGCACT 1410 ATTGCACT 2347 TTGCACT 3284 hsa-miR-301bCAGUGCAAUGAUAUUGUCAAAGC 474 CATTGCACT 1411 ATTGCACT 2348 TTGCACT 3285hsa-miR-302a UAAGUGCUUCCAUGUUUUGGUGA 475 GAAGCACTT 1412 AAGCACTT 2349AGCACTT 3286 hsa-miR-302a* ACUUAAACGUGGAUGUACUUGCU 476 ACGTTTAAG 1413CGTTTAAG 2350 GTTTAAG 3287 hsa-miR-302b UAAGUGCUUCCAUGUUUUAGUAG 477GAAGCACTT 1414 AAGCACTT 2351 AGCACTT 3288 hsa-miR-302b*ACUUUAACAUGGAAGUGCUUUC 478 ATGTTAAAG 1415 TGTTAAAG 2352 GTTAAAG 3289hsa-miR-302c UAAGUGCUUCCAUGUUUCAGUGG 479 GAAGCACTT 1416 AAGCACTT 2353AGCACTT 3290 hsa-miR-302c* UUUAACAUGGGGGUACCUGCUG 480 CCATGTTAA 1417CATGTTAA 2354 ATGTTAA 3291 hsa-miR-302d UAAGUGCUUCCAUGUUUGAGUGU 481GAAGCACTT 1418 AAGCACTT 2355 AGCACTT 3292 hsa-miR-302d*ACUUUAACAUGGAGGCACUUGC 482 ATGTTAAAG 1419 TGTTAAAG 2356 GTTAAAG 3293hsa-miR-302e UAAGUGCUUCCAUGCUU 483 GAAGCACTT 1420 AAGCACTT 2357 AGCACTT3294 hsa-miR-302f UAAUUGCUUCCAUGUUU 484 GAAGCAATT 1421 AAGCAATT 2358AGCAATT 3295 hsa-miR-30a UGUAAACAUCCUCGACUGGAAG 485 GATGTTTAC 1422ATGTTTAC 2359 TGTTTAC 3296 hsa-miR-30a* CUUUCAGUCGGAUGUUUGCAGC 486CGACTGAAA 1423 GACTGAAA 2360 ACTGAAA 3297 hsa-miR-30bUGUAAACAUCCUACACUCAGCU 487 GATGTTTAC 1424 ATGTTTAC 2361 TGTTTAC 3298hsa-miR-30b* CUGGGAGGUGGAUGUUUACUUC 488 CACCTCCCA 1425 ACCTCCCA 2362CCTCCCA 3299 hsa-miR-30c UGUAAACAUCCUACACUCUCAGC 489 GATGTTTAC 1426ATGTTTAC 2363 TGTTTAC 3300 hsa-miR-30c-1* CUGGGAGAGGGUUGUUUACUCC 490CCTCTCCCA 1427 CTCTCCCA 2364 TCTCCCA 3301 hsa-miR-30c-2*CUGGGAGAAGGCUGUUUACUCU 491 CTTCTCCCA 1428 TTCTCCCA 2365 TCTCCCA 3302hsa-miR-30d UGUAAACAUCCCCGACUGGAAG 492 GATGTTTAC 1429 ATGTTTAC 2366TGTTTAC 3303 hsa-miR-30d* CUUUCAGUCAGAUGUUUGCUGC 493 TGACTGAAA 1430GACTGAAA 2367 ACTGAAA 3304 hsa-miR-30e UGUAAACAUCCUUGACUGGAAG 494GATGTTTAC 1431 ATGTTTAC 2368 TGTTTAC 3305 hsa-miR-30e*CUUUCAGUCGGAUGUUUACAGC 495 CGACTGAAA 1432 GACTGAAA 2369 ACTGAAA 3306hsa-miR-31 AGGCAAGAUGCUGGCAUAGCU 496 CATCTTGCC 1433 ATCTTGCC 2370TCTTGCC 3307 hsa-miR-31* UGCUAUGCCAACAUAUUGCCAU 497 TGGCATAGC 1434GGCATAGC 2371 GCATAGC 3308 hsa-miR-32 UAUUGCACAUUACUAAGUUGCA 498ATGTGCAAT 1435 TGTGCAAT 2372 GTGCAAT 3309 hsa-miR-32*CAAUUUAGUGUGUGUGAUAUUU 499 CACTAAATT 1436 ACTAAATT 2373 CTAAATT 3310hsa-miR-320a AAAAGCUGGGUUGAGAGGGCGA 500 CCCAGCTTT 1437 CCAGCTTT 2374CAGCTTT 3311 hsa-miR-320b AAAAGCUGGGUUGAGAGGGCAA 501 CCCAGCTTT 1438CCAGCTTT 2375 CAGCTTT 3312 hsa-miR-320c AAAAGCUGGGUUGAGAGGGU 502CCCAGCTTT 1439 CCAGCTTT 2376 CAGCTTT 3313 hsa-miR-320dAAAAGCUGGGUUGAGAGGA 503 CCCAGCTTT 1440 CCAGCTTT 2377 CAGCTTT 3314hsa-miR-323-3p CACAUUACACGGUCGACCUCU 504 GTGTAATGT 1441 TGTAATGT 2378GTAATGT 3315 hsa-miR-323-5p AGGUGGUCCGUGGCGCGUUCGC 505 CGGACCACC 1442GGACCACC 2379 GACCACC 3316 hsa-miR-324-3p ACUGCCCCAGGUGCUGCUGG 506CTGGGGCAG 1443 TGGGGCAG 2380 GGGGCAG 3317 hsa-miR-324-5pCGCAUCCCCUAGGGCAUUGGUGU 507 AGGGGATGC 1444 GGGGATGC 2381 GGGATGC 3318hsa-miR-325 CCUAGUAGGUGUCCAGUAAGUGU 508 ACCTACTAG 1445 CCTACTAG 2382CTACTAG 3319 hsa-miR-326 CCUCUGGGCCCUUCCUCCAG 509 GGCCCAGAG 1446GCCCAGAG 2383 CCCAGAG 3320 hsa-miR-328 CUGGCCCUCUCUGCCCUUCCGU 510AGAGGGCCA 1447 GAGGGCCA 2384 AGGGCCA 3321 hsa-miR-329AACACACCUGGUUAACCUCUUU 511 CAGGTGTGT 1448 AGGTGTGT 2385 GGTGTGT 3322hsa-miR-330-3p GCAAAGCACACGGCCUGCAGAGA 512 TGTGCTTTG 1449 GTGCTTTG 2386TGCTTTG 3323 hsa-miR-330-5p UCUCUGGGCCUGUGUCUUAGGC 513 GGCCCAGAG 1450GCCCAGAG 2387 CCCAGAG 3324 hsa-miR-331-3p GCCCCUGGGCCUAUCCUAGAA 514GCCCAGGGG 1451 CCCAGGGG 2388 CCAGGGG 3325 hsa-miR-331-5pCUAGGUAUGGUCCCAGGGAUCC 515 CCATACCTA 1452 CATACCTA 2389 ATACCTA 3326hsa-miR-335 UCAAGAGCAAUAACGAAAAAUGU 516 TTGCTCTTG 1453 TGCTCTTG 2390GCTCTTG 3327 hsa-miR-335* UUUUUCAUUAUUGCUCCUGACC 517 TAATGAAAA 1454AATGAAAA 2391 ATGAAAA 3328 hsa-miR-337-3p CUCCUAUAUGAUGCCUUUCUUC 518CATATAGGA 1455 ATATAGGA 2392 TATAGGA 3329 hsa-miR-337-5pGAACGGCUUCAUACAGGAGUU 519 GAAGCCGTT 1456 AAGCCGTT 2393 AGCCGTT 3330hsa-miR-338-3p UCCAGCAUCAGUGAUUUUGUUG 520 TGATGCTGG 1457 GATGCTGG 2394ATGCTGG 3331 hsa-miR-338-5p AACAAUAUCCUGGUGCUGAGUG 521 GGATATTGT 1458GATATTGT 2395 ATATTGT 3332 hsa-miR-339-3p UGAGCGCCUCGACGACAGAGCCG 522GAGGCGCTC 1459 AGGCGCTC 2396 GGCGCTC 3333 hsa-miR-339-5pUCCCUGUCCUCCAGGAGCUCACG 523 AGGACAGGG 1460 GGACAGGG 2397 GACAGGG 3334hsa-miR-33a GUGCAUUGUAGUUGCAUUGCA 524 TACAATGCA 1461 ACAATGCA 2398CAATGCA 3335 hsa-miR-33a* CAAUGUUUCCACAGUGCAUCAC 525 GGAAACATT 1462GAAACATT 2399 AAACATT 3336 hsa-miR-33b GUGCAUUGCUGUUGCAUUGC 526AGCAATGCA 1463 GCAATGCA 2400 CAATGCA 3337 hsa-miR-33b*CAGUGCCUCGGCAGUGCAGCCC 527 CGAGGCACT 1464 GAGGCACT 2401 AGGCACT 3338hsa-miR-340 UUAUAAAGCAAUGAGACUGAUU 528 TGCTTTATA 1465 GCTTTATA 2402CTTTATA 3339 hsa-miR-340* UCCGUCUCAGUUACUUUAUAGC 529 CTGAGACGG 1466TGAGACGG 2403 GAGACGG 3340 hsa-miR-342-3p UCUCACACAGAAAUCGCACCCGU 530CTGTGTGAG 1467 TGTGTGAG 2404 GTGTGAG 3341 hsa-miR-342-5pAGGGGUGCUAUCUGUGAUUGA 531 TAGCACCCC 1468 AGCACCCC 2405 GCACCCC 3342hsa-miR-345 GCUGACUCCUAGUCCAGGGCUC 532 AGGAGTCAG 1469 GGAGTCAG 2406GAGTCAG 3343 hsa-miR-346 UGUCUGCCCGCAUGCCUGCCUCU 533 CGGGCAGAC 1470GGGCAGAC 2407 GGCAGAC 3344 hsa-miR-34a UGGCAGUGUCUUAGCUGGUUGU 534GACACTGCC 1471 ACACTGCC 2408 CACTGCC 3345 hsa-miR-34a*CAAUCAGCAAGUAUACUGCCCU 535 TTGCTGATT 1472 TGCTGATT 2409 GCTGATT 3346hsa-miR-34b CAAUCACUAACUCCACUGCCAU 536 TTAGTGATT 1473 TAGTGATT 2410AGTGATT 3347 hsa-miR-34b* UAGGCAGUGUCAUUAGCUGAUUG 537 ACACTGCCT 1474CACTGCCT 2411 ACTGCCT 3348 hsa-miR-34c-3p AAUCACUAACCACACGGCCAGG 538GTTAGTGAT 1475 TTAGTGAT 2412 TAGTGAT 3349 hsa-miR-34c-5pAGGCAGUGUAGUUAGCUGAUUGC 539 TACACTGCC 1476 ACACTGCC 2413 CACTGCC 3350hsa-miR-361-3p UCCCCCAGGUGUGAUUCUGAUUU 540 ACCTGGGGG 1477 CCTGGGGG 2414CTGGGGG 3351 hsa-miR-361-5p UUAUCAGAAUCUCCAGGGGUAC 541 ATTCTGATA 1478TTCTGATA 2415 TCTGATA 3352 hsa-miR-362-3p AACACACCUAUUCAAGGAUUCA 542TAGGTGTGT 1479 AGGTGTGT 2416 GGTGTGT 3353 hsa-miR-362-5pAAUCCUUGGAACCUAGGUGUGAGU 543 TCCAAGGAT 1480 CCAAGGAT 2417 CAAGGAT 3354hsa-miR-363 AAUUGCACGGUAUCCAUCUGUA 544 CCGTGCAAT 1481 CGTGCAAT 2418GTGCAAT 3355 hsa-miR-363* CGGGUGGAUCACGAUGCAAUUU 545 GATCCACCC 1482ATCCACCC 2419 TCCACCC 3356 hsa-miR-365 UAAUGCCCCUAAAAAUCCUUAU 546AGGGGCATT 1483 GGGGCATT 2420 GGGCATT 3357 hsa-miR-367AAUUGCACUUUAGCAAUGGUGA 547 AAGTGCAAT 1484 AGTGCAAT 2421 GTGCAAT 3358hsa-miR-367* ACUGUUGCUAAUAUGCAACUCU 548 TAGCAACAG 1485 AGCAACAG 2422GCAACAG 3359 hsa-miR-369-3p AAUAAUACAUGGUUGAUCUUU 549 ATGTATTAT 1486TGTATTAT 2423 GTATTAT 3360 hsa-miR-369-5p AGAUCGACCGUGUUAUAUUCGC 550CGGTCGATC 1487 GGTCGATC 2424 GTCGATC 3361 hsa-miR-370GCCUGCUGGGGUGGAACCUGGU 551 CCCAGCAGG 1488 CCAGCAGG 2425 CAGCAGG 3362hsa-miR-371-3p AAGUGCCGCCAUCUUUUGAGUGU 552 GGCGGCACT 1489 GCGGCACT 2426CGGCACT 3363 hsa-miR-371-5p ACUCAAACUGUGGGGGCACU 553 CAGTTTGAG 1490AGTTTGAG 2427 GTTTGAG 3364 hsa-miR-372 AAAGUGCUGCGACAUUUGAGCGU 554GCAGCACTT 1491 CAGCACTT 2428 AGCACTT 3365 hsa-miR-373GAAGUGCUUCGAUUUUGGGGUGU 555 GAAGCACTT 1492 AAGCACTT 2429 AGCACTT 3366hsa-miR-373* ACUCAAAAUGGGGGCGCUUUCC 556 CATTTTGAG 1493 ATTTTGAG 2430TTTTGAG 3367 hsa-miR-374a UUAUAAUACAACCUGAUAAGUG 557 TGTATTATA 1494GTATTATA 2431 TATTATA 3368 hsa-miR-374a* CUUAUCAGAUUGUAUUGUAAUU 558ATCTGATAA 1495 TCTGATAA 2432 CTGATAA 3369 hsa-miR-374bAUAUAAUACAACCUGCUAAGUG 559 TGTATTATA 1496 GTATTATA 2433 TATTATA 3370hsa-miR-374b* CUUAGCAGGUUGUAUUAUCAUU 560 ACCTGCTAA 1497 CCTGCTAA 2434CTGCTAA 3371 hsa-miR-375 UUUGUUCGUUCGGCUCGCGUGA 561 AACGAACAA 1498ACGAACAA 2435 CGAACAA 3372 hsa-miR-376a AUCAUAGAGGAAAAUCCACGU 562CCTCTATGA 1499 CTCTATGA 2436 TCTATGA 3373 hsa-miR-376a*GUAGAUUCUCCUUCUAUGAGUA 563 GAGAATCTA 1500 AGAATCTA 2437 GAATCTA 3374hsa-miR-376b AUCAUAGAGGAAAAUCCAUGUU 564 CCTCTATGA 1501 CTCTATGA 2438TCTATGA 3375 hsa-miR-376c AACAUAGAGGAAAUUCCACGU 565 CCTCTATGT 1502CTCTATGT 2439 TCTATGT 3376 hsa-miR-377 AUCACACAAAGGCAACUUUUGU 566TTTGTGTGA 1503 TTGTGTGA 2440 TGTGTGA 3377 hsa-miR-377*AGAGGUUGCCCUUGGUGAAUUC 567 GGCAACCTC 1504 GCAACCTC 2441 CAACCTC 3378hsa-miR-378 ACUGGACUUGGAGUCAGAAGG 568 CAAGTCCAG 1505 AAGTCCAG 2442AGTCCAG 3379 hsa-miR-378* CUCCUGACUCCAGGUCCUGUGU 569 GAGTCAGGA 1506AGTCAGGA 2443 GTCAGGA 3380 hsa-miR-379 UGGUAGACUAUGGAACGUAGG 570TAGTCTACC 1507 AGTCTACC 2444 GTCTACC 3381 hsa-miR-379*UAUGUAACAUGGUCCACUAACU 571 ATGTTACAT 1508 TGTTACAT 2445 GTTACAT 3382hsa-miR-380 UAUGUAAUAUGGUCCACAUCUU 572 ATATTACAT 1509 TATTACAT 2446ATTACAT 3383 hsa-miR-380* UGGUUGACCAUAGAACAUGCGC 573 TGGTCAACC 1510GGTCAACC 2447 GTCAACC 3384 hsa-miR-381 UAUACAAGGGCAAGCUCUCUGU 574CCCTTGTAT 1511 CCTTGTAT 2448 CTTGTAT 3385 hsa-miR-382GAAGUUGUUCGUGGUGGAUUCG 575 GAACAACTT 1512 AACAACTT 2449 ACAACTT 3386hsa-miR-383 AGAUCAGAAGGUGAUUGUGGCU 576 CTTCTGATC 1513 TTCTGATC 2450TCTGATC 3387 hsa-miR-384 AUUCCUAGAAAUUGUUCAUA 577 TTCTAGGAA 1514TCTAGGAA 2451 CTAGGAA 3388 hsa-miR-409-3p GAAUGUUGCUCGGUGAACCCCU 578AGCAACATT 1515 GCAACATT 2452 CAACATT 3389 hsa-miR-409-5pAGGUUACCCGAGCAACUUUGCAU 579 CGGGTAACC 1516 GGGTAACC 2453 GGTAACC 3390hsa-miR-410 AAUAUAACACAGAUGGCCUGU 580 GTGTTATAT 1517 TGTTATAT 2454GTTATAT 3391 hsa-miR-411 UAGUAGACCGUAUAGCGUACG 581 CGGTCTACT 1518GGTCTACT 2455 GTCTACT 3392 hsa-miR-411* UAUGUAACACGGUCCACUAACC 582GTGTTACAT 1519 TGTTACAT 2456 GTTACAT 3393 hsa-miR-412ACUUCACCUGGUCCACUAGCCGU 583 CAGGTGAAG 1520 AGGTGAAG 2457 GGTGAAG 3394hsa-miR-421 AUCAACAGACAUUAAUUGGGCGC 584 GTCTGTTGA 1521 TCTGTTGA 2458CTGTTGA 3395 hsa-miR-422a ACUGGACUUAGGGUCAGAAGGC 585 TAAGTCCAG 1522AAGTCCAG 2459 AGTCCAG 3396 hsa-miR-423-3p AGCUCGGUCUGAGGCCCCUCAGU 586AGACCGAGC 1523 GACCGAGC 2460 ACCGAGC 3397 hsa-miR-423-5pUGAGGGGCAGAGAGCGAGACUUU 587 CTGCCCCTC 1524 TGCCCCTC 2461 GCCCCTC 3398hsa-miR-424 CAGCAGCAAUUCAUGUUUUGAA 588 ATTGCTGCT 1525 TTGCTGCT 2462TGCTGCT 3399 hsa-miR-424* CAAAACGUGAGGCGCUGCUAU 589 TCACGTTTT 1526CACGTTTT 2463 ACGTTTT 3400 hsa-miR-425 AAUGACACGAUCACUCCCGUUGA 590TCGTGTCAT 1527 CGTGTCAT 2464 GTGTCAT 3401 hsa-miR-425*AUCGGGAAUGUCGUGUCCGCCC 591 CATTCCCGA 1528 ATTCCCGA 2465 TTCCCGA 3402hsa-miR-429 UAAUACUGUCUGGUAAAACCGU 592 GACAGTATT 1529 ACAGTATT 2466CAGTATT 3403 hsa-miR-431 UGUCUUGCAGGCCGUCAUGCA 593 CTGCAAGAC 1530TGCAAGAC 2467 GCAAGAC 3404 hsa-miR-431* CAGGUCGUCUUGCAGGGCUUCU 594AGACGACCT 1531 GACGACCT 2468 ACGACCT 3405 hsa-miR-432UCUUGGAGUAGGUCAUUGGGUGG 595 TACTCCAAG 1532 ACTCCAAG 2469 CTCCAAG 3406hsa-miR-432* CUGGAUGGCUCCUCCAUGUCU 596 AGCCATCCA 1533 GCCATCCA 2470CCATCCA 3407 hsa-miR-433 AUCAUGAUGGGCUCCUCGGUGU 597 CCATCATGA 1534CATCATGA 2471 ATCATGA 3408 hsa-miR-448 UUGCAUAUGUAGGAUGUCCCAU 598ACATATGCA 1535 CATATGCA 2472 ATATGCA 3409 hsa-miR-449aUGGCAGUGUAUUGUUAGCUGGU 599 TACACTGCC 1536 ACACTGCC 2473 CACTGCC 3410hsa-miR-449b AGGCAGUGUAUUGUUAGCUGGC 600 TACACTGCC 1537 ACACTGCC 2474CACTGCC 3411 hsa-miR-450a UUUUGCGAUGUGUUCCUAAUAU 601 CATCGCAAA 1538ATCGCAAA 2475 TCGCAAA 3412 hsa-miR-450b-3p UUGGGAUCAUUUUGCAUCCAUA 602ATGATCCCA 1539 TGATCCCA 2476 GATCCCA 3413 hsa-miR-450b-5pUUUUGCAAUAUGUUCCUGAAUA 603 TATTGCAAA 1540 ATTGCAAA 2477 TTGCAAA 3414hsa-miR-451 AAACCGUUACCAUUACUGAGUU 604 GTAACGGTT 1541 TAACGGTT 2478AACGGTT 3415 hsa-miR-452 AACUGUUUGCAGAGGAAACUGA 605 GCAAACAGT 1542CAAACAGT 2479 AAACAGT 3416 hsa-miR-452* CUCAUCUGCAAAGAAGUAAGUG 606TGCAGATGA 1543 GCAGATGA 2480 CAGATGA 3417 hsa-miR-453AGGUUGUCCGUGGUGAGUUCGCA 607 CGGACAACC 1544 GGACAACC 2481 GACAACC 3418hsa-miR-454 UAGUGCAAUAUUGCUUAUAGGGU 608 TATTGCACT 1545 ATTGCACT 2482TTGCACT 3419 hsa-miR-454* ACCCUAUCAAUAUUGUCUCUGC 609 TTGATAGGG 1546TGATAGGG 2483 GATAGGG 3420 hsa-miR-455-3p GCAGUCCAUGGGCAUAUACAC 610CATGGACTG 1547 ATGGACTG 2484 TGGACTG 3421 hsa-miR-455-5pUAUGUGCCUUUGGACUACAUCG 611 AAGGCACAT 1548 AGGCACAT 2485 GGCACAT 3422hsa-miR-483-3p UCACUCCUCUCCUCCCGUCUU 612 AGAGGAGTG 1549 GAGGAGTG 2486AGGAGTG 3423 hsa-miR-483-5p AAGACGGGAGGAAAGAAGGGAG 613 CTCCCGTCT 1550TCCCGTCT 2487 CCCGTCT 3424 hsa-miR-484 UCAGGCUCAGUCCCCUCCCGAU 614CTGAGCCTG 1551 TGAGCCTG 2488 GAGCCTG 3425 hsa-miR-485-3pGUCAUACACGGCUCUCCUCUCU 615 CGTGTATGA 1552 GTGTATGA 2489 TGTATGA 3426hsa-miR-485-5p AGAGGCUGGCCGUGAUGAAUUC 616 GCCAGCCTC 1553 CCAGCCTC 2490CAGCCTC 3427 hsa-miR-486-3p CGGGGCAGCUCAGUACAGGAU 617 AGCTGCCCC 1554GCTGCCCC 2491 CTGCCCC 3428 hsa-miR-486-5p UCCUGUACUGAGCUGCCCCGAG 618CAGTACAGG 1555 AGTACAGG 2492 GTACAGG 3429 hsa-miR-487aAAUCAUACAGGGACAUCCAGUU 619 CTGTATGAT 1556 TGTATGAT 2493 GTATGAT 3430hsa-miR-487b AAUCGUACAGGGUCAUCCACUU 620 CTGTACGAT 1557 TGTACGAT 2494GTACGAT 3431 hsa-miR-488 UUGAAAGGCUAUUUCUUGGUC 621 AGCCTTTCA 1558GCCTTTCA 2495 CCTTTCA 3432 hsa-miR-488* CCCAGAUAAUGGCACUCUCAA 622ATTATCTGG 1559 TTATCTGG 2496 TATCTGG 3433 hsa-miR-489GUGACAUCACAUAUACGGCAGC 623 GTGATGTCA 1560 TGATGTCA 2497 GATGTCA 3434hsa-miR-490-3p CAACCUGGAGGACUCCAUGCUG 624 CTCCAGGTT 1561 TCCAGGTT 2498CCAGGTT 3435 hsa-miR-490-5p CCAUGGAUCUCCAGGUGGGU 625 AGATCCATG 1562GATCCATG 2499 ATCCATG 3436 hsa-miR-491-3p CUUAUGCAAGAUUCCCUUCUAC 626CTTGCATAA 1563 TTGCATAA 2500 TGCATAA 3437 hsa-miR-491-5pAGUGGGGAACCCUUCCAUGAGG 627 GTTCCCCAC 1564 TTCCCCAC 2501 TCCCCAC 3438hsa-miR-492 AGGACCUGCGGGACAAGAUUCUU 628 CGCAGGTCC 1565 GCAGGTCC 2502CAGGTCC 3439 hsa-miR-493 UGAAGGUCUACUGUGUGCCAGG 629 TAGACCTTC 1566AGACCTTC 2503 GACCTTC 3440 hsa-miR-493* UUGUACAUGGUAGGCUUUCAUU 630CCATGTACA 1567 CATGTACA 2504 ATGTACA 3441 hsa-miR-494UGAAACAUACACGGGAAACCUC 631 GTATGTTTC 1568 TATGTTTC 2505 ATGTTTC 3442hsa-miR-495 AAACAAACAUGGUGCACUUCUU 632 ATGTTTGTT 1569 TGTTTGTT 2506GTTTGTT 3443 hsa-miR-496 UGAGUAUUACAUGGCCAAUCUC 633 GTAATACTC 1570TAATACTC 2507 AATACTC 3444 hsa-miR-497 CAGCAGCACACUGUGGUUUGU 634TGTGCTGCT 1571 GTGCTGCT 2508 TGCTGCT 3445 hsa-miR-497*CAAACCACACUGUGGUGUUAGA 635 GTGTGGTTT 1572 TGTGGTTT 2509 GTGGTTT 3446hsa-miR-498 UUUCAAGCCAGGGGGCGUUUUUC 636 TGGCTTGAA 1573 GGCTTGAA 2510GCTTGAA 3447 hsa-miR-499-3p AACAUCACAGCAAGUCUGUGCU 637 CTGTGATGT 1574TGTGATGT 2511 GTGATGT 3448 hsa-miR-499-5p UUAAGACUUGCAGUGAUGUUU 638CAAGTCTTA 1575 AAGTCTTA 2512 AGTCTTA 3449 hsa-miR-500UAAUCCUUGCUACCUGGGUGAGA 639 GCAAGGATT 1576 CAAGGATT 2513 AAGGATT 3450hsa-miR-500* AUGCACCUGGGCAAGGAUUCUG 640 CCAGGTGCA 1577 CAGGTGCA 2514AGGTGCA 3451 hsa-miR-501-3p AAUGCACCCGGGCAAGGAUUCU 641 CGGGTGCAT 1578GGGTGCAT 2515 GGTGCAT 3452 hsa-miR-501-5p AAUCCUUUGUCCCUGGGUGAGA 642ACAAAGGAT 1579 CAAAGGAT 2516 AAAGGAT 3453 hsa-miR-502-3pAAUGCACCUGGGCAAGGAUUCA 643 CAGGTGCAT 1580 AGGTGCAT 2517 GGTGCAT 3454hsa-miR-502-5p AUCCUUGCUAUCUGGGUGCUA 644 TAGCAAGGA 1581 AGCAAGGA 2518GCAAGGA 3455 hsa-miR-503 UAGCAGCGGGAACAGUUCUGCAG 645 CCCGCTGCT 1582CCGCTGCT 2519 CGCTGCT 3456 hsa-miR-504 AGACCCUGGUCUGCACUCUAUC 646ACCAGGGTC 1583 CCAGGGTC 2520 CAGGGTC 3457 hsa-miR-505CGUCAACACUUGCUGGUUUCCU 647 AGTGTTGAC 1584 GTGTTGAC 2521 TGTTGAC 3458hsa-miR-505* GGGAGCCAGGAAGUAUUGAUGU 648 CCTGGCTCC 1585 CTGGCTCC 2522TGGCTCC 3459 hsa-miR-506 UAAGGCACCCUUCUGAGUAGA 649 GGGTGCCTT 1586GGTGCCTT 2523 GTGCCTT 3460 hsa-miR-507 UUUUGCACCUUUUGGAGUGAA 650AGGTGCAAA 1587 GGTGCAAA 2524 GTGCAAA 3461 hsa-miR-508-3pUGAUUGUAGCCUUUUGGAGUAGA 651 GCTACAATC 1588 CTACAATC 2525 TACAATC 3462hsa-miR-508-5p UACUCCAGAGGGCGUCACUCAUG 652 CTCTGGAGT 1589 TCTGGAGT 2526CTGGAGT 3463 hsa-miR-509-3-5p UACUGCAGACGUGGCAAUCAUG 653 GTCTGCAGT 1590TCTGCAGT 2527 CTGCAGT 3464 hsa-miR-509-3p UGAUUGGUACGUCUGUGGGUAG 654GTACCAATC 1591 TACCAATC 2528 ACCAATC 3465 hsa-miR-509-5pUACUGCAGACAGUGGCAAUCA 655 GTCTGCAGT 1592 TCTGCAGT 2529 CTGCAGT 3466hsa-miR-510 UACUCAGGAGAGUGGCAAUCAC 656 CTCCTGAGT 1593 TCCTGAGT 2530CCTGAGT 3467 hsa-miR-511 GUGUCUUUUGCUCUGCAGUCA 657 CAAAAGACA 1594AAAAGACA 2531 AAAGACA 3468 hsa-miR-512-3p AAGUGCUGUCAUAGCUGAGGUC 658GACAGCACT 1595 ACAGCACT 2532 CAGCACT 3469 hsa-miR-512-5pCACUCAGCCUUGAGGGCACUUUC 659 AGGCTGAGT 1596 GGCTGAGT 2533 GCTGAGT 3470hsa-miR-513a-3p UAAAUUUCACCUUUCUGAGAAGG 660 GTGAAATTT 1597 TGAAATTT 2534GAAATTT 3471 hsa-miR-513a-5p UUCACAGGGAGGUGUCAU 661 TCCCTGTGA 1598CCCTGTGA 2535 CCTGTGA 3472 hsa-miR-513b UUCACAAGGAGGUGUCAUUUAU 662TCCTTGTGA 1599 CCTTGTGA 2536 CTTGTGA 3473 hsa-miR-513cUUCUCAAGGAGGUGUCGUUUAU 663 TCCTTGAGA 1600 CCTTGAGA 2537 CTTGAGA 3474hsa-miR-514 AUUGACACUUCUGUGAGUAGA 664 AAGTGTCAA 1601 AGTGTCAA 2538GTGTCAA 3475 hsa-miR-515-3p GAGUGCCUUCUUUUGGAGCGUU 665 GAAGGCACT 1602AAGGCACT 2539 AGGCACT 3476 hsa-miR-515-5p UUCUCCAAAAGAAAGCACUUUCUG 666TTTTGGAGA 1603 TTTGGAGA 2540 TTGGAGA 3477 hsa-miR-516a-3pUGCUUCCUUUCAGAGGGU 667 AAAGGAAGC 1604 AAGGAAGC 2541 AGGAAGC 3478hsa-miR-516a-5p UUCUCGAGGAAAGAAGCACUUUC 668 TCCTCGAGA 1605 CCTCGAGA 2542CTCGAGA 3479 hsa-miR-516b AUCUGGAGGUAAGAAGCACUUU 669 ACCTCCAGA 1606CCTCCAGA 2543 CTCCAGA 3480 hsa-miR-517* CCUCUAGAUGGAAGCACUGUCU 670CATCTAGAG 1607 ATCTAGAG 2544 TCTAGAG 3481 hsa-miR-517aAUCGUGCAUCCCUUUAGAGUGU 671 GATGCACGA 1608 ATGCACGA 2545 TGCACGA 3482hsa-miR-517b UCGUGCAUCCCUUUAGAGUGUU 672 GGATGCACG 1609 GATGCACG 2546ATGCACG 3483 hsa-miR-517c AUCGUGCAUCCUUUUAGAGUGU 673 GATGCACGA 1610ATGCACGA 2547 TGCACGA 3484 hsa-miR-518a-3p GAAAGCGCUUCCCUUUGCUGGA 674AAGCGCTTT 1611 AGCGCTTT 2548 GCGCTTT 3485 hsa-miR-518bCAAAGCGCUCCCCUUUAGAGGU 675 GAGCGCTTT 1612 AGCGCTTT 2549 GCGCTTT 3486hsa-miR-518c CAAAGCGCUUCUCUUUAGAGUGU 676 AAGCGCTTT 1613 AGCGCTTT 2550GCGCTTT 3487 hsa-miR-518c* UCUCUGGAGGGAAGCACUUUCUG 677 CCTCCAGAG 1614CTCCAGAG 2551 TCCAGAG 3488 hsa-miR-518d-3p CAAAGCGCUUCCCUUUGGAGC 678AAGCGCTTT 1615 AGCGCTTT 2552 GCGCTTT 3489 hsa-miR-518d-5pCUCUAGAGGGAAGCACUUUCUG 679 CCCTCTAGA 1616 CCTCTAGA 2553 CTCTAGA 3490hsa-miR-518e AAAGCGCUUCCCUUCAGAGUG 680 GAAGCGCTT 1617 AAGCGCTT 2554AGCGCTT 3491 hsa-miR-518f GAAAGCGCUUCUCUUUAGAGG 681 AAGCGCTTT 1618AGCGCTTT 2555 GCGCTTT 3492 hsa-miR-518f* CUCUAGAGGGAAGCACUUUCUC 682CCCTCTAGA 1619 CCTCTAGA 2556 CTCTAGA 3493 hsa-miR-519aAAAGUGCAUCCUUUUAGAGUGU 683 GATGCACTT 1620 ATGCACTT 2557 TGCACTT 3494hsa-miR-519a* CUCUAGAGGGAAGCGCUUUCUG 684 CCCTCTAGA 1621 CCTCTAGA 2558CTCTAGA 3495 hsa-miR-519b-3p AAAGUGCAUCCUUUUAGAGGUU 685 GATGCACTT 1622ATGCACTT 2559 TGCACTT 3496 hsa-miR-519c-3p AAAGUGCAUCUUUUUAGAGGAU 686GATGCACTT 1623 ATGCACTT 2560 TGCACTT 3497 hsa-miR-519dCAAAGUGCCUCCCUUUAGAGUG 687 AGGCACTTT 1624 GGCACTTT 2561 GCACTTT 3498hsa-miR-519e AAGUGCCUCCUUUUAGAGUGUU 688 GGAGGCACT 1625 GAGGCACT 2562AGGCACT 3499 hsa-miR-519e* UUCUCCAAAAGGGAGCACUUUC 689 TTTTGGAGA 1626TTTGGAGA 2563 TTGGAGA 3500 hsa-miR-520a-3p AAAGUGCUUCCCUUUGGACUGU 690GAAGCACTT 1627 AAGCACTT 2564 AGCACTT 3501 hsa-miR-520a-5pCUCCAGAGGGAAGUACUUUCU 691 CCCTCTGGA 1628 CCTCTGGA 2565 CTCTGGA 3502hsa-miR-520b AAAGUGCUUCCUUUUAGAGGG 692 GAAGCACTT 1629 AAGCACTT 2566AGCACTT 3503 hsa-miR-520c-3p AAAGUGCUUCCUUUUAGAGGGU 693 GAAGCACTT 1630AAGCACTT 2567 AGCACTT 3504 hsa-miR-520d-3p AAAGUGCUUCUCUUUGGUGGGU 694GAAGCACTT 1631 AAGCACTT 2568 AGCACTT 3505 hsa-miR-520d-5pCUACAAAGGGAAGCCCUUUC 695 CCCTTTGTA 1632 CCTTTGTA 2569 CTTTGTA 3506hsa-miR-520e AAAGUGCUUCCUUUUUGAGGG 696 GAAGCACTT 1633 AAGCACTT 2570AGCACTT 3507 hsa-miR-520f AAGUGCUUCCUUUUAGAGGGUU 697 GGAAGCACT 1634GAAGCACT 2571 AAGCACT 3508 hsa-miR-520g ACAAAGUGCUUCCCUUUAGAGUGU 698AGCACTTTG 1635 GCACTTTG 2572 CACTTTG 3509 hsa-miR-520hACAAAGUGCUUCCCUUUAGAGU 699 AGCACTTTG 1636 GCACTTTG 2573 CACTTTG 3510hsa-miR-521 AACGCACUUCCCUUUAGAGUGU 700 GAAGTGCGT 1637 AAGTGCGT 2574AGTGCGT 3511 hsa-miR-522 AAAAUGGUUCCCUUUAGAGUGU 701 GAACCATTT 1638AACCATTT 2575 ACCATTT 3512 hsa-miR-523 GAACGCGCUUCCCUAUAGAGGGU 702AAGCGCGTT 1639 AGCGCGTT 2576 GCGCGTT 3513 hsa-miR-524-3pGAAGGCGCUUCCCUUUGGAGU 703 AAGCGCCTT 1640 AGCGCCTT 2577 GCGCCTT 3514hsa-miR-524-5p CUACAAAGGGAAGCACUUUCUC 704 CCCTTTGTA 1641 CCTTTGTA 2578CTTTGTA 3515 hsa-miR-525-3p GAAGGCGCUUCCCUUUAGAGCG 705 AAGCGCCTT 1642AGCGCCTT 2579 GCGCCTT 3516 hsa-miR-525-5p CUCCAGAGGGAUGCACUUUCU 706CCCTCTGGA 1643 CCTCTGGA 2580 CTCTGGA 3517 hsa-miR-526bCUCUUGAGGGAAGCACUUUCUGU 707 CCCTCAAGA 1644 CCTCAAGA 2581 CTCAAGA 3518hsa-miR-526b* GAAAGUGCUUCCUUUUAGAGGC 708 AAGCACTTT 1645 AGCACTTT 2582GCACTTT 3519 hsa-miR-527 CUGCAAAGGGAAGCCCUUUC 709 CCCTTTGCA 1646CCTTTGCA 2583 CTTTGCA 3520 hsa-miR-532-3p CCUCCCACACCCAAGGCUUGCA 710GTGTGGGAG 1647 TGTGGGAG 2584 GTGGGAG 3521 hsa-miR-532-5pCAUGCCUUGAGUGUAGGACCGU 711 TCAAGGCAT 1648 CAAGGCAT 2585 AAGGCAT 3522hsa-miR-539 GGAGAAAUUAUCCUUGGUGUGU 712 TAATTTCTC 1649 AATTTCTC 2586ATTTCTC 3523 hsa-miR-541 UGGUGGGCACAGAAUCUGGACU 713 GTGCCCACC 1650TGCCCACC 2587 GCCCACC 3524 hsa-miR-541* AAAGGAUUCUGCUGUCGGUCCCACU 714AGAATCCTT 1651 GAATCCTT 2588 AATCCTT 3525 hsa-miR-542-3pUGUGACAGAUUGAUAACUGAAA 715 ATCTGTCAC 1652 TCTGTCAC 2589 CTGTCAC 3526hsa-miR-542-5p UCGGGGAUCAUCAUGUCACGAGA 716 TGATCCCCG 1653 GATCCCCG 2590ATCCCCG 3527 hsa-miR-543 AAACAUUCGCGGUGCACUUCUU 717 GCGAATGTT 1654CGAATGTT 2591 GAATGTT 3528 hsa-miR-544 AUUCUGCAUUUUUAGCAAGUUC 718AATGCAGAA 1655 ATGCAGAA 2592 TGCAGAA 3529 hsa-miR-545UCAGCAAACAUUUAUUGUGUGC 719 TGTTTGCTG 1656 GTTTGCTG 2593 TTTGCTG 3530hsa-miR-545* UCAGUAAAUGUUUAUUAGAUGA 720 CATTTACTG 1657 ATTTACTG 2594TTTACTG 3531 hsa-miR-548a-3p CAAAACUGGCAAUUACUUUUGC 721 GCCAGTTTT 1658CCAGTTTT 2595 CAGTTTT 3532 hsa-miR-548a-5p AAAAGUAAUUGCGAGUUUUACC 722AATTACTTT 1659 ATTACTTT 2596 TTACTTT 3533 hsa-miR-548b-3pCAAGAACCUCAGUUGCUUUUGU 723 GAGGTTCTT 1660 AGGTTCTT 2597 GGTTCTT 3534hsa-miR-548b-5p AAAAGUAAUUGUGGUUUUGGCC 724 AATTACTTT 1661 ATTACTTT 2598TTACTTT 3535 hsa-miR-548c-3p CAAAAAUCUCAAUUACUUUUGC 725 GAGATTTTT 1662AGATTTTT 2599 GATTTTT 3536 hsa-miR-548c-5p AAAAGUAAUUGCGGUUUUUGCC 726AATTACTTT 1663 ATTACTTT 2600 TTACTTT 3537 hsa-miR-548d-3pCAAAAACCACAGUUUCUUUUGC 727 GTGGTTTTT 1664 TGGTTTTT 2601 GGTTTTT 3538hsa-miR-548d-5p AAAAGUAAUUGUGGUUUUUGCC 728 AATTACTTT 1665 ATTACTTT 2602TTACTTT 3539 hsa-miR-548e AAAAACUGAGACUACUUUUGCA 729 CTCAGTTTT 1666TCAGTTTT 2603 CAGTTTT 3540 hsa-miR-548f AAAAACUGUAAUUACUUUU 730TACAGTTTT 1667 ACAGTTTT 2604 CAGTTTT 3541 hsa-miR-548gAAAACUGUAAUUACUUUUGUAC 731 TTACAGTTT 1668 TACAGTTT 2605 ACAGTTT 3542hsa-miR-548h AAAAGUAAUCGCGGUUUUUGUC 732 GATTACTTT 1669 ATTACTTT 2606TTACTTT 3543 hsa-miR-548i AAAAGUAAUUGCGGAUUUUGCC 733 AATTACTTT 1670ATTACTTT 2607 TTACTTT 3544 hsa-miR-548j AAAAGUAAUUGCGGUCUUUGGU 734AATTACTTT 1671 ATTACTTT 2608 TTACTTT 3545 hsa-miR-548kAAAAGUACUUGCGGAUUUUGCU 735 AAGTACTTT 1672 AGTACTTT 2609 GTACTTT 3546hsa-miR-548l AAAAGUAUUUGCGGGUUUUGUC 736 AAATACTTT 1673 AATACTTT 2610ATACTTT 3547 hsa-miR-548m CAAAGGUAUUUGUGGUUUUUG 737 AATACCTTT 1674ATACCTTT 2611 TACCTTT 3548 hsa-miR-548n CAAAAGUAAUUGUGGAUUUUGU 738ATTACTTTT 1675 TTACTTTT 2612 TACTTTT 3549 hsa-miR-548oCCAAAACUGCAGUUACUUUUGC 739 GCAGTTTTG 1676 CAGTTTTG 2613 AGTTTTG 3550hsa-miR-548p UAGCAAAAACUGCAGUUACUUU 740 GTTTTTGCT 1677 TTTTTGCT 2614TTTTGCT 3551 hsa-miR-549 UGACAACUAUGGAUGAGCUCU 741 ATAGTTGTC 1678TAGTTGTC 2615 AGTTGTC 3552 hsa-miR-550 AGUGCCUGAGGGAGUAAGAGCCC 742CTCAGGCAC 1679 TCAGGCAC 2616 CAGGCAC 3553 hsa-miR-550*UGUCUUACUCCCUCAGGCACAU 743 GAGTAAGAC 1680 AGTAAGAC 2617 GTAAGAC 3554hsa-miR-551a GCGACCCACUCUUGGUUUCCA 744 AGTGGGTCG 1681 GTGGGTCG 2618TGGGTCG 3555 hsa-miR-551b GCGACCCAUACUUGGUUUCAG 745 TATGGGTCG 1682ATGGGTCG 2619 TGGGTCG 3556 hsa-miR-551b* GAAAUCAAGCGUGGGUGAGACC 746GCTTGATTT 1683 CTTGATTT 2620 TTGATTT 3557 hsa-miR-552AACAGGUGACUGGUUAGACAA 747 GTCACCTGT 1684 TCACCTGT 2621 CACCTGT 3558hsa-miR-553 AAAACGGUGAGAUUUUGUUUU 748 TCACCGTTT 1685 CACCGTTT 2622ACCGTTT 3559 hsa-miR-554 GCUAGUCCUGACUCAGCCAGU 749 CAGGACTAG 1686AGGACTAG 2623 GGACTAG 3560 hsa-miR-555 AGGGUAAGCUGAACCUCUGAU 750AGCTTACCC 1687 GCTTACCC 2624 CTTACCC 3561 hsa-miR-556-3pAUAUUACCAUUAGCUCAUCUUU 751 ATGGTAATA 1688 TGGTAATA 2625 GGTAATA 3562hsa-miR-556-5p GAUGAGCUCAUUGUAAUAUGAG 752 TGAGCTCAT 1689 GAGCTCAT 2626AGCTCAT 3563 hsa-miR-557 GUUUGCACGGGUGGGCCUUGUCU 753 CCGTGCAAA 1690CGTGCAAA 2627 GTGCAAA 3564 hsa-miR-558 UGAGCUGCUGUACCAAAAU 754 CAGCAGCTC1691 AGCAGCTC 2628 GCAGCTC 3565 hsa-miR-559 UAAAGUAAAUAUGCACCAAAA 755ATTTACTTT 1692 TTTACTTT 2629 TTACTTT 3566 hsa-miR-561CAAAGUUUAAGAUCCUUGAAGU 756 TTAAACTTT 1693 TAAACTTT 2630 AAACTTT 3567hsa-miR-562 AAAGUAGCUGUACCAUUUGC 757 CAGCTACTT 1694 AGCTACTT 2631GCTACTT 3568 hsa-miR-563 AGGUUGACAUACGUUUCCC 758 ATGTCAACC 1695 TGTCAACC2632 GTCAACC 3569 hsa-miR-564 AGGCACGGUGUCAGCAGGC 759 CACCGTGCC 1696ACCGTGCC 2633 CCGTGCC 3570 hsa-miR-566 GGGCGCCUGUGAUCCCAAC 760 ACAGGCGCC1697 CAGGCGCC 2634 AGGCGCC 3571 hsa-miR-567 AGUAUGUUCUUCCAGGACAGAAC 761AGAACATAC 1698 GAACATAC 2635 AACATAC 3572 hsa-miR-568AUGUAUAAAUGUAUACACAC 762 ATTTATACA 1699 TTTATACA 2636 TTATACA 3573hsa-miR-569 AGUUAAUGAAUCCUGGAAAGU 763 TTCATTAAC 1700 TCATTAAC 2637CATTAC 3574 hsa-miR-570 CGAAAACAGCAAUUACCUUUGC 764 GCTGTTTTC 1701CTGTTTTC 2638 TGTTTTC 3575 hsa-miR-571 UGAGUUGGCCAUCUGAGUGAG 765GGCCAACTC 1702 GCCAACTC 2639 CCAACTC 3576 hsa-miR-572GUCCGCUCGGCGGUGGCCCA 766 CCGAGCGGA 1703 CGAGCGGA 2640 GAGCGGA 3577hsa-miR-573 CUGAAGUGAUGUGUAACUGAUCAG 767 ATCACTTCA 1704 TCACTTCA 2641CACTTCA 3578 hsa-miR-574-3p CACGCUCAUGCACACACCCACA 768 CATGAGCGT 1705ATGAGCGT 2642 TGAGCGT 3579 hsa-miR-574-5p UGAGUGUGUGUGUGUGAGUGUGU 769CACACACTC 1706 ACACACTC 2643 CACACTC 3580 hsa-miR-575GAGCCAGUUGGACAGGAGC 770 CAACTGGCT 1707 AACTGGCT 2644 ACTGGCT 3581hsa-miR-576-3p AAGAUGUGGAAAAAUUGGAAUC 771 TCCACATCT 1708 CCACATCT 2645CACATCT 3582 hsa-miR-576-5p AUUCUAAUUUCUCCACGUCUUU 772 AAATTAGAA 1709AATTAGAA 2646 ATTAGAA 3583 hsa-miR-577 UAGAUAAAAUAUUGGUACCUG 773ATTTTATCT 1710 TTTTATCT 2647 TTTATCT 3584 hsa-miR-578CUUCUUGUGCUCUAGGAUUGU 774 GCACAAGAA 1711 CACAAGAA 2648 ACAAGAA 3585hsa-miR-579 UUCAUUUGGUAUAAACCGCGAUU 775 ACCAAATGA 1712 CCAAATGA 2649CAAATGA 3586 hsa-miR-580 UUGAGAAUGAUGAAUCAUUAGG 776 TCATTCTCA 1713CATTCTCA 2650 ATTCTCA 3587 hsa-miR-581 UCUUGUGUUCUCUAGAUCAGU 777GAACACAAG 1714 AACACAAG 2651 ACACAAG 3588 hsa-miR-582-3pUAACUGGUUGAACAACUGAACC 778 CAACCAGTT 1715 AACCAGTT 2652 ACCAGTT 3589hsa-miR-582-5p UUACAGUUGUUCAACCAGUUACU 779 ACAACTGTA 1716 CAACTGTA 2653AACTGTA 3590 hsa-miR-583 CAAAGAGGAAGGUCCCAUUAC 780 TTCCTCTTT 1717TCCTCTTT 2654 CCTCTTT 3591 hsa-miR-584 UUAUGGUUUGCCUGGGACUGAG 781CAAACCATA 1718 AAACCATA 2655 AACCATA 3592 hsa-miR-585UGGGCGUAUCUGUAUGCUA 782 GATACGCCC 1719 ATACGCCC 2656 TACGCCC 3593hsa-miR-586 UAUGCAUUGUAUUUUUAGGUCC 783 ACAATGCAT 1720 CAATGCAT 2657AATGCAT 3594 hsa-miR-587 UUUCCAUAGGUGAUGAGUCAC 784 CCTATGGAA 1721CTATGGAA 2658 TATGGA 3595 hsa-miR-588 UUGGCCACAAUGGGUUAGAAC 785TTGTGGCCA 1722 TGTGGCCA 2659 GTGGCCA 3596 hsa-miR-589UGAGAACCACGUCUGCUCUGAG 786 GTGGTTCTC 1723 TGGTTCTC 2660 GGTTCTC 3597hsa-miR-589* UCAGAACAAAUGCCGGUUCCCAGA 787 TTTGTTCTG 1724 TTGTTCTG 2661TGTTCTG 3598 hsa-miR-590-3p UAAUUUUAUGUAUAAGCUAGU 788 CATAAAATT 1725ATAAAATT 2662 TAAAATT 3599 hsa-miR-590-5p GAGCUUAUUCAUAAAAGUGCAG 789GAATAAGCT 1726 AATAAGCT 2663 ATAAGCT 3600 hsa-miR-591AGACCAUGGGUUCUCAUUGU 790 CCCATGGTC 1727 CCATGGTC 2664 CATGGTC 3601hsa-miR-592 UUGUGUCAAUAUGCGAUGAUGU 791 ATTGACACA 1728 TTGACACA 2665TGACACA 3602 hsa-miR-593 UGUCUCUGCUGGGGUUUCU 792 AGCAGAGAC 1729 GCAGAGAC2666 CAGAGAC 3603 hsa-miR-593* AGGCACCAGCCAGGCAUUGCUCAGC 793 GCTGGTGCC1730 CTGGTGCC 2667 TGGTGCC 3604 hsa-miR-595 GAAGUGUGCCGUGGUGUGUCU 794GGCACACTT 1731 GCACACTT 2668 CACACTT 3605 hsa-miR-596AAGCCUGCCCGGCUCCUCGGG 795 GGGCAGGCT 1732 GGCAGGCT 2669 GCAGGCT 3606hsa-miR-597 UGUGUCACUCGAUGACCACUGU 796 GAGTGACAC 1733 AGTGACAC 2670GTGACAC 3607 hsa-miR-598 UACGUCAUCGUUGUCAUCGUCA 797 CGATGACGT 1734GATGACGT 2671 ATGACGT 3608 hsa-miR-599 GUUGUGUCAGUUUAUCAAAC 798CTGACACAA 1735 TGACACAA 2672 GACACAA 3609 hsa-miR-600ACUUACAGACAAGAGCCUUGCUC 799 GTCTGTAAG 1736 TCTGTAAG 2673 CTGTAAG 3610hsa-miR-601 UGGUCUAGGAUUGUUGGAGGAG 800 TCCTAGACC 1737 CCTAGACC 2674CTAGACC 3611 hsa-miR-602 GACACGGGCGACAGCUGCGGCCC 801 CGCCCGTGT 1738GCCCGTGT 2675 CCCGTGT 3612 hsa-miR-603 CACACACUGCAAUUACUUUUGC 802GCAGTGTGT 1739 CAGTGTGT 2676 AGTGTGT 3613 hsa-miR-604AGGCUGCGGAAUUCAGGAC 803 TCCGCAGCC 1740 CCGCAGCC 2677 CGCAGCC 3614hsa-miR-605 UAAAUCCCAUGGUGCCUUCUCCU 804 ATGGGATTT 1741 TGGGATTT 2678GGGATTT 3615 hsa-miR-606 AAACUACUGAAAAUCAAAGAU 805 TCAGTAGTT 1742CAGTAGTT 2679 AGTAGTT 3616 hsa-miR-607 GUUCAAAUCCAGAUCUAUAAC 806GGATTTGAA 1743 GATTTGAA 2680 ATTTGAA 3617 hsa-miR-608AGGGGUGGUGUUGGGACAGCUCCGU 807 CACCACCCC 1744 ACCACCCC 2681 CCACCCC 3618hsa-miR-609 AGGGUGUUUCUCUCAUCUCU 808 GAAACACCC 1745 AAACACCC 2682AACACCC 3619 hsa-miR-610 UGAGCUAAAUGUGUGCUGGGA 809 ATTTAGCTC 1746TTTAGCTC 2683 TTAGCTC 3620 hsa-miR-611 GCGAGGACCCCUCGGGGUCUGAC 810GGGTCCTCG 1747 GGTCCTCG 2684 GTCCTCG 3621 hsa-miR-612GCUGGGCAGGGCUUCUGAGCUCCUU 811 CCTGCCCAG 1748 CTGCCCAG 2685 TGCCCAG 3622hsa-miR-613 AGGAAUGUUCCUUCUUUGCC 812 GAACATTCC 1749 AACATTCC 2686ACATTCC 3623 hsa-miR-614 GAACGCCUGUUCUUGCCAGGUGG 813 ACAGGCGTT 1750CAGGCGTT 2687 AGGCGTT 3624 hsa-miR-615-3p UCCGAGCCUGGGUCUCCCUCUU 814CAGGCTCGG 1751 AGGCTCGG 2688 GGCTCGG 3625 hsa-miR-615-5pGGGGGUCCCCGGUGCUCGGAUC 815 GGGGACCCC 1752 GGGACCCC 2689 GGACCCC 3626hsa-miR-616 AGUCAUUGGAGGGUUUGAGCAG 816 TCCAATGAC 1753 CCAATGAC 2690CAATGAC 3627 hsa-miR-616* ACUCAAAACCCUUCAGUGACUU 817 GGTTTTGAG 1754GTTTTGAG 2691 TTTTGAG 3628 hsa-miR-617 AGACUUCCCAUUUGAAGGUGGC 818TGGGAAGTC 1755 GGGAAGTC 2692 GGAAGTC 3629 hsa-miR-618AAACUCUACUUGUCCUUCUGAGU 819 AGTAGAGTT 1756 GTAGAGTT 2693 TAGAGTT 3630hsa-miR-619 GACCUGGACAUGUUUGUGCCCAGU 820 TGTCCAGGT 1757 GTCCAGGT 2694TCCAGGT 3631 hsa-miR-620 AUGGAGAUAGAUAUAGAAAU 821 CTATCTCCA 1758TATCTCCA 2695 ATCTCCA 3632 hsa-miR-621 GGCUAGCAACAGCGCUUACCU 822GTTGCTAGC 1759 TTGCTAGC 2696 TGCTAGC 3633 hsa-miR-622ACAGUCUGCUGAGGUUGGAGC 823 AGCAGACTG 1760 GCAGACTG 2697 CAGACTG 3634hsa-miR-623 AUCCCUUGCAGGGGCUGUUGGGU 824 TGCAAGGGA 1761 GCAAGGGA 2698CAAGGGA 3635 hsa-miR-624 CACAAGGUAUUGGUAUUACCU 825 ATACCTTGT 1762TACCTTGT 2699 ACCTTGT 3636 hsa-miR-624* UAGUACCAGUACCUUGUGUUCA 826ACTGGTACT 1763 CTGGTACT 2700 TGGTACT 3637 hsa-miR-625AGGGGGAAAGUUCUAUAGUCC 827 CTTTCCCCC 1764 TTTCCCCC 2701 TTCCCCC 3638hsa-miR-625* GACUAUAGAACUUUCCCCCUCA 828 TTCTATAGT 1765 TCTATAGT 2702CTATAGT 3639 hsa-miR-626 AGCUGUCUGAAAAUGUCUU 829 TCAGACAGC 1766 CAGACAGC2703 AGACAGC 3640 hsa-miR-627 GUGAGUCUCUAAGAAAAGAGGA 830 AGAGACTCA 1767GAGACTCA 2704 AGACTCA 3641 hsa-miR-628-3p UCUAGUAAGAGUGGCAGUCGA 831TCTTACTAG 1768 CTTACTAG 2705 TTACTAG 3642 hsa-miR-628-5pAUGCUGACAUAUUUACUAGAGG 832 ATGTCAGCA 1769 TGTCAGCA 2706 GTCAGCA 3643hsa-miR-629 UGGGUUUACGUUGGGAGAACU 833 CGTAAACCC 1770 GTAAACCC 2707TAAACCC 3644 hsa-miR-629* GUUCUCCCAACGUAAGCCCAGC 834 TTGGGAGAA 1771TGGGAGAA 2708 GGGAGAA 3645 hsa-miR-630 AGUAUUCUGUACCAGGGAAGGU 835ACAGAATAC 1772 CAGAATAC 2709 AGAATAC 3646 hsa-miR-631AGACCUGGCCCAGACCUCAGC 836 GGCCAGGTC 1773 GCCAGGTC 2710 CCAGGTC 3647hsa-miR-632 GUGUCUGCUUCCUGUGGGA 837 AAGCAGACA 1774 AGCAGACA 2711 GCAGACA3648 hsa-miR-633 CUAAUAGUAUCUACCACAAUAAA 838 ATACTATTA 1775 TACTATTA2712 ACTATTA 3649 hsa-miR-634 AACCAGCACCCCAACUUUGGAC 839 GGTGCTGGT 1776GTGCTGGT 2713 TGCTGGT 3650 hsa-miR-635 ACUUGGGCACUGAAACAAUGUCC 840GTGCCCAAG 1777 TGCCCAAG 2714 GCCCAAG 3651 hsa-miR-636UGUGCUUGCUCGUCCCGCCCGCA 841 AGCAAGCAC 1778 GCAAGCAC 2715 CAAGCAC 3652hsa-miR-637 ACUGGGGGCUUUCGGGCUCUGCGU 842 AGCCCCCAG 1779 GCCCCCAG 2716CCCCCAG 3653 hsa-miR-638 AGGGAUCGCGGGCGGGUGGCGGCCU 843 CGCGATCCC 1780GCGATCCC 2717 CGATCCC 3654 hsa-miR-639 AUCGCUGCGGUUGCGAGCGCUGU 844CCGCAGCGA 1781 CGCAGCGA 2718 GCAGCGA 3655 hsa-miR-640AUGAUCCAGGAACCUGCCUCU 845 CCTGGATCA 1782 CTGGATCA 2719 TGGATCA 3656hsa-miR-641 AAAGACAUAGGAUAGAGUCACCUC 846 CTATGTCTT 1783 TATGTCTT 2720ATGTCTT 3657 hsa-miR-642 GUCCCUCUCCAAAUGUGUCUUG 847 GGAGAGGGA 1784GAGAGGGA 2721 AGAGGGA 3658 hsa-miR-643 ACUUGUAUGCUAGCUCAGGUAG 848GCATACAAG 1785 CATACAAG 2722 ATACAAG 3659 hsa-miR-644AGUGUGGCUUUCUUAGAGC 849 AAGCCACAC 1786 AGCCACAC 2723 GCCACAC 3660hsa-miR-645 UCUAGGCUGGUACUGCUGA 850 CCAGCCTAG 1787 CAGCCTAG 2724 AGCCTAG3661 hsa-miR-646 AAGCAGCUGCCUCUGAGGC 851 GCAGCTGCT 1788 CAGCTGCT 2725AGCTGCT 3662 hsa-miR-647 GUGGCUGCACUCACUUCCUUC 852 GTGCAGCCA 1789TGCAGCCA 2726 GCAGCCA 3663 hsa-miR-648 AAGUGUGCAGGGCACUGGU 853 CTGCACACT1790 TGCACACT 2727 GCACACT 3664 hsa-miR-649 AAACCUGUGUUGUUCAAGAGUC 854ACACAGGTT 1791 CACAGGTT 2728 ACAGGTT 3665 hsa-miR-650AGGAGGCAGCGCUCUCAGGAC 855 GCTGCCTCC 1792 CTGCCTCC 2729 TGCCTCC 3666hsa-miR-651 UUUAGGAUAAGCUUGACUUUUG 856 TTATCCTAA 1793 TATCCTAA 2730ATCCTAA 3667 hsa-miR-652 AAUGGCGCCACUAGGGUUGUG 857 TGGCGCCAT 1794GGCGCCAT 2731 GCGCCAT 3668 hsa-miR-653 GUGUUGAAACAAUCUCUACUG 858GTTTCAACA 1795 TTTCAACA 2732 TTCAACA 3669 hsa-miR-654-3pUAUGUCUGCUGACCAUCACCUU 859 AGCAGACAT 1796 GCAGACAT 2733 CAGACAT 3670hsa-miR-654-5p UGGUGGGCCGCAGAACAUGUGC 860 CGGCCCACC 1797 GGCCCACC 2734GCCCACC 3671 hsa-miR-655 AUAAUACAUGGUUAACCUCUUU 861 CATGTATTA 1798ATGTATTA 2735 TGTATTA 3672 hsa-miR-656 AAUAUUAUACAGUCAACCUCU 862GTATAATAT 1799 TATAATAT 2736 ATAATAT 3673 hsa-miR-657GGCAGGUUCUCACCCUCUCUAGG 863 AGAACCTGC 1800 GAACCTGC 2737 AACCTGC 3674hsa-miR-658 GGCGGAGGGAAGUAGGUCCGUUGGU 864 TCCCTCCGC 1801 CCCTCCGC 2738CCTCCGC 3675 hsa-miR-659 CUUGGUUCAGGGAGGGUCCCCA 865 CTGAACCAA 1802TGAACCAA 2739 GAACCAA 3676 hsa-miR-660 UACCCAUUGCAUAUCGGAGUUG 866GCAATGGGT 1803 CAATGGGT 2740 AATGGGT 3677 hsa-miR-661UGCCUGGGUCUCUGGCCUGCGCGU 867 GACCCAGGC 1804 ACCCAGGC 2741 CCCAGGC 3678hsa-miR-662 UCCCACGUUGUGGCCCAGCAG 868 CAACGTGGG 1805 AACGTGGG 2742ACGTGGG 3679 hsa-miR-663 AGGCGGGGCGCCGCGGGACCGC 869 CGCCCCGCC 1806GCCCCGCC 2743 CCCCGCC 3680 hsa-miR-663b GGUGGCCCGGCCGUGCCUGAGG 870CCGGGCCAC 1807 CGGGCCAC 2744 GGGCCAC 3681 hsa-miR-664UAUUCAUUUAUCCCCAGCCUACA 871 TAAATGAAT 1808 AAATGAAT 2745 AATGAAT 3682hsa-miR-664* ACUGGCUAGGGAAAAUGAUUGGAU 872 CCTAGCCAG 1809 CTAGCCAG 2746TAGCCAG 3683 hsa-miR-665 ACCAGGAGGCUGAGGCCCCU 873 GCCTCCTGG 1810CCTCCTGG 2747 CTCCTGG 3684 hsa-miR-668 UGUCACUCGGCUCGGCCCACUAC 874CCGAGTGAC 1811 CGAGTGAC 2748 GAGTGAC 3685 hsa-miR-671-3pUCCGGUUCUCAGGGCUCCACC 875 GAGAACCGG 1812 AGAACCGG 2749 GAACCGG 3686hsa-miR-671-5p AGGAAGCCCUGGAGGGGCUGGAG 876 AGGGCTTCC 1813 GGGCTTCC 2750GGCTTCC 3687 hsa-miR-675 UGGUGCGGAGAGGGCCCACAGUG 877 CTCCGCACC 1814TCCGCACC 2751 CCGCACC 3688 hsa-miR-675b CUGUAUGCCCUCACCGCUCA 878GGGCATACA 1815 GGCATACA 2752 GCATACA 3689 hsa-miR-7UGGAAGACUAGUGAUUUUGUUGU 879 TAGTCTTCC 1816 AGTCTTCC 2753 GTCTTCC 3690hsa-miR-7-1* CAACAAAUCACAGUCUGCCAUA 880 TGATTTGTT 1817 GATTTGTT 2754ATTTGTT 3691 hsa-miR-7-2* CAACAAAUCCCAGUCUACCUAA 881 GGATTTGTT 1818GATTTGTT 2755 ATTTGTT 3692 hsa-miR-708 AAGGAGCUUACAAUCUAGCUGGG 882TAAGCTCCT 1819 AAGCTCCT 2756 AGCTCCT 3693 hsa-miR-708*CAACUAGACUGUGAGCUUCUAG 883 AGTCTAGTT 1820 GTCTAGTT 2757 TCTAGTT 3694hsa-miR-720 UCUCGCUGGGGCCUCCA 884 CCCAGCGAG 1821 CCAGCGAG 2758 CAGCGAG3695 hsa-miR-744 UGCGGGGCUAGGGCUAACAGCA 885 TAGCCCCGC 1822 AGCCCCGC 2759GCCCCGC 3696 hsa-miR-744* CUGUUGCCACUAACCUCAACCU 886 GTGGCAACA 1823TGGCAACA 2760 GGCAACA 3697 hsa-miR-758 UUUGUGACCUGGUCCACUAACC 887AGGTCACAA 1824 GGTCACAA 2761 GTCACAA 3698 hsa-miR-760CGGCUCUGGGUCUGUGGGGA 888 CCCAGAGCC 1825 CCAGAGCC 2762 CAGAGCC 3699hsa-miR-765 UGGAGGAGAAGGAAGGUGAUG 889 TTCTCCTCC 1826 TCTCCTCC 2763CTCCTCC 3700 hsa-miR-766 ACUCCAGCCCCACAGCCUCAGC 890 GGGCTGGAG 1827GGCTGGAG 2764 GCTGGAG 3701 hsa-miR-767-3p UCUGCUCAUACCCCAUGGUUUCU 891TATGAGCAG 1828 ATGAGCAG 2765 TGAGCAG 3702 hsa-miR-767-5pUGCACCAUGGUUGUCUGAGCAUG 892 CCATGGTGC 1829 CATGGTGC 2766 ATGGTGC 3703hsa-miR-769-3p CUGGGAUCUCCGGGGUCUUGGUU 893 GAGATCCCA 1830 AGATCCCA 2767GATCCCA 3704 hsa-miR-769-5p UGAGACCUCUGGGUUCUGAGCU 894 AGAGGTCTC 1831GAGGTCTC 2768 AGGTCTC 3705 hsa-miR-770-5p UCCAGUACCACGUGUCAGGGCCA 895TGGTACTGG 1832 GGTACTGG 2769 GTACTGG 3706 hsa-miR-802CAGUAACAAAGAUUCAUCCUUGU 896 TTTGTTACT 1833 TTGTTACT 2770 TGTTACT 3707hsa-miR-873 GCAGGAACUUGUGAGUCUCCU 897 AAGTTCCTG 1834 AGTTCCTG 2771GTTCCTG 3708 hsa-miR-874 CUGCCCUGGCCCGAGGGACCGA 898 GCCAGGGCA 1835CCAGGGCA 2772 CAGGGCA 3709 hsa-miR-875-3p CCUGGAAACACUGAGGUUGUG 899TGTTTCCAG 1836 GTTTCCAG 2773 TTTCCAG 3710 hsa-miR-875-5pUAUACCUCAGUUUUAUCAGGUG 900 CTGAGGTAT 1837 TGAGGTAT 2774 GAGGTAT 3711hsa-miR-876-3p UGGUGGUUUACAAAGUAAUUCA 901 TAAACCACC 1838 AAACCACC 2775AACCACC 3712 hsa-miR-876-5p UGGAUUUCUUUGUGAAUCACCA 902 AAGAAATCC 1839AGAAATCC 2776 GAAATCC 3713 hsa-miR-877 GUAGAGGAGAUGGCGCAGGG 903TCTCCTCTA 1840 CTCCTCTA 2777 TCCTCTA 3714 hsa-miR-877*UCCUCUUCUCCCUCCUCCCAG 904 GAGAAGAGG 1841 AGAAGAGG 2778 GAAGAGG 3715hsa-miR-885-3p AGGCAGCGGGGUGUAGUGGAUA 905 CCCGCTGCC 1842 CCGCTGCC 2779CGCTGCC 3716 hsa-miR-885-5p UCCAUUACACUACCCUGCCUCU 906 GTGTAATGG 1843TGTAATGG 2780 GTAATGG 3717 hsa-miR-886-3p CGCGGGUGCUUACUGACCCUU 907AGCACCCGC 1844 GCACCCGC 2781 CACCCGC 3718 hsa-miR-886-5pCGGGUCGGAGUUAGCUCAAGCGG 908 CTCCGACCC 1845 TCCGACCC 2782 CCGACCC 3719hsa-miR-887 GUGAACGGGCGCCAUCCCGAGG 909 GCCCGTTCA 1846 CCCGTTCA 2783CCGTTCA 3720 hsa-miR-888 UACUCAAAAAGCUGUCAGUCA 910 TTTTTGAGT 1847TTTTGAGT 2784 TTTGAGT 3721 hsa-miR-888* GACUGACACCUCUUUGGGUGAA 911GGTGTCAGT 1848 GTGTCAGT 2785 TGTCAGT 3722 hsa-miR-889UUAAUAUCGGACAACCAUUGU 912 CCGATATTA 1849 CGATATTA 2786 GATATTA 3723hsa-miR-890 UACUUGGAAAGGCAUCAGUUG 913 TTTCCAAGT 1850 TTCCAAGT 2787TCCAAGT 3724 hsa-miR-891a UGCAACGAACCUGAGCCACUGA 914 GTTCGTTGC 1851TTCGTTGC 2788 TCGTTGC 3725 hsa-miR-891b UGCAACUUACCUGAGUCAUUGA 915GTAAGTTGC 1852 TAAGTTGC 2789 AAGTTGC 3726 hsa-miR-892aCACUGUGUCCUUUCUGCGUAG 916 GGACACAGT 1853 GACACAGT 2790 ACACAGT 3727hsa-miR-892b CACUGGCUCCUUUCUGGGUAGA 917 GGAGCCAGT 1854 GAGCCAGT 2791AGCCAGT 3728 hsa-miR-9 UCUUUGGUUAUCUAGCUGUAUGA 918 TAACCAAAG 1855AACCAAAG 2792 ACCAAAG 3729 hsa-miR-9* AUAAAGCUAGAUAACCGAAAGU 919CTAGCTTTA 1856 TAGCTTTA 2793 AGCTTTA 3730 hsa-miR-920GGGGAGCUGUGGAAGCAGUA 920 ACAGCTCCC 1857 CAGCTCCC 2794 AGCTCCC 3731hsa-miR-921 CUAGUGAGGGACAGAACCAGGAUUC 921 CCCTCACTA 1858 CCTCACTA 2795CTCACTA 3732 hsa-miR-922 GCAGCAGAGAAUAGGACUACGUC 922 TCTCTGCTG 1859CTCTGCTG 2796 TCTGCTG 3733 hsa-miR-923 GUCAGCGGAGGAAAAGAAACU 923CTCCGCTGA 1860 TCCGCTGA 2797 CCGCTGA 3734 hsa-miR-924AGAGUCUUGUGAUGUCUUGC 924 ACAAGACTC 1861 CAAGACTC 2798 AAGACTC 3735hsa-miR-92a UAUUGCACUUGUCCCGGCCUGU 925 AAGTGCAAT 1862 AGTGCAAT 2799GTGCAAT 3736 hsa-miR-92a-1* AGGUUGGGAUCGGUUGCAAUGCU 926 ATCCCAACC 1863TCCCAACC 2800 CCCAACC 3737 hsa-miR-92a-2* GGGUGGGGAUUUGUUGCAUUAC 927ATCCCCACC 1864 TCCCCACC 2801 CCCCACC 3738 hsa-miR-92bUAUUGCACUCGUCCCGGCCUCC 928 GAGTGCAAT 1865 AGTGCAAT 2802 GTGCAAT 3739hsa-miR-92b* AGGGACGGGACGCGGUGCAGUG 929 TCCCGTCCC 1866 CCCGTCCC 2803CCGTCCC 3740 hsa-miR-93 CAAAGUGCUGUUCGUGCAGGUAG 930 CAGCACTTT 1867AGCACTTT 2804 GCACTTT 3741 hsa-miR-93* ACUGCUGAGCUAGCACUUCCCG 931GCTCAGCAG 1868 CTCAGCAG 2805 TCAGCAG 3742 hsa-miR-933UGUGCGCAGGGAGACCUCUCCC 932 CCTGCGCAC 1869 CTGCGCAC 2806 TGCGCAC 3743hsa-miR-934 UGUCUACUACUGGAGACACUGG 933 GTAGTAGAC 1870 TAGTAGAC 2807AGTAGAC 3744 hsa-miR-935 CCAGUUACCGCUUCCGCUACCGC 934 CGGTAACTG 1871GGTAACTG 2808 GTAACTG 3745 hsa-miR-936 ACAGUAGAGGGAGGAAUCGCAG 935CCTCTACTG 1872 CTCTACTG 2809 TCTACTG 3746 hsa-miR-937AUCCGCGCUCUGACUCUCUGCC 936 GAGCGCGGA 1873 AGCGCGGA 2810 GCGCGGA 3747hsa-miR-938 UGCCCUUAAAGGUGAACCCAGU 937 TTTAAGGGC 1874 TTAAGGGC 2811TAAGGGC 3748 hsa-miR-939 UGGGGAGCUGAGGCUCUGGGGGUG 938 CAGCTCCCC 1875AGCTCCCC 2812 GCTCCCC 3749 hsa-miR-940 AAGGCAGGGCCCCCGCUCCCC 939GCCCTGCCT 1876 CCCTGCCT 2813 CCTGCCT 3750 hsa-miR-941CACCCGGCUGUGUGCACAUGUGC 940 CAGCCGGGT 1877 AGCCGGGT 2814 GCCGGGT 3751hsa-miR-942 UCUUCUCUGUUUUGGCCAUGUG 941 ACAGAGAAG 1878 CAGAGAAG 2815AGAGAAG 3752 hsa-miR-943 CUGACUGUUGCCGUCCUCCAG 942 CAACAGTCA 1879AACAGTCA 2816 ACAGTCA 3753 hsa-miR-944 AAAUUAUUGUACAUCGGAUGAG 943ACAATAATT 1880 CAATAATT 2817 AATAATT 3754 hsa-miR-95UUCAACGGGUAUUUAUUGAGCA 944 ACCCGTTGA 1881 CCCGTTGA 2818 CCGTTGA 3755hsa-miR-96 UUUGGCACUAGCACAUUUUUGCU 945 TAGTGCCAA 1882 AGTGCCAA 2819GTGCCAA 3756 hsa-miR-96* AAUCAUGUGCAGUGCCAAUAUG 946 GCACATGAT 1883CACATGAT 2820 ACATGAT 3757 hsa-miR-98 UGAGGUAGUAAGUUGUAUUGUU 947TACTACCTC 1884 ACTACCTC 2821 CTACCTC 3758 hsa-miR-99aAACCCGUAGAUCCGAUCUUGUG 948 TCTACGGGT 1885 CTACGGGT 2822 TACGGGT 3759hsa-miR-99a* CAAGCUCGCUUCUAUGGGUCUG 949 AGCGAGCTT 1886 GCGAGCTT 2823CGAGCTT 3760 hsa-miR-99b CACCCGUAGAACCGACCUUGCG 950 TCTACGGGT 1887CTACGGGT 2824 TACGGGT 3761 hsa-miR-99b* CAAGCUCGUGUCUGUGGGUCCG 951CACGAGCTT 1888 ACGAGCTT 2825 CGAGCTT 3762 hsv1-miR-H1UGGAAGGACGGGAAGUGGAAG 952 CGTCCTTCC 1889 GTCCTTCC 2826 TCCTTCC 3763hsv1-miR-H2-3p CCUGAGCCAGGGACGAGUGCGACU 953 CTGGCTCAG 1890 TGGCTCAG 2827GGCTCAG 3764 hsv1-miR-H2-5p UCGCACGCGCCCGGCACAGACU 954 GCGCGTGCG 1891CGCGTGCG 2828 GCGTGCG 3765 hsv1-miR-H3 CUGGGACUGUGCGGUUGGGA 955ACAGTCCCA 1892 CAGTCCCA 2829 AGTCCCA 3766 hsv1-miR-H4-3pCUUGCCUGUCUAACUCGCUAGU 956 GACAGGCAA 1893 ACAGGCAA 2830 CAGGCAA 3767hsv1-miR-H4-5p GGUAGAGUUUGACAGGCAAGCA 957 AAACTCTAC 1894 AACTCTAC 2831ACTCTAC 3768 hsv1-miR-H5 GUCAGAGAUCCAAACCCUCCGG 958 GATCTCTGA 1895ATCTCTGA 2832 TCTCTGA 3769 hsv1-miR-H6 CACUUCCCGUCCUUCCAUCCC 959ACGGGAAGT 1896 CGGGAAGT 2833 GGGAAGT 3770 kshv-miR-K12-1AUUACAGGAAACUGGGUGUAAGC 960 TTCCTGTAA 1897 TCCTGTAA 2834 CCTGTAA 3771kshv-miR-K12-10a UAGUGUUGUCCCCCCGAGUGGC 961 GACAACACT 1898 ACAACACT 2835CAACACT 3772 kshv-miR-K12-10b UGGUGUUGUCCCCCCGAGUGGC 962 GACAACACC 1899ACAACACC 2836 CAACACC 3773 kshv-miR-K12-11 UUAAUGCUUAGCCUGUGUCCGA 963TAAGCATTA 1900 AAGCATTA 2837 AGCATTA 3774 kshv-miR-K12-12ACCAGGCCACCAUUCCUCUCCG 964 GTGGCCTGG 1901 TGGCCTGG 2838 GGCCTGG 3775kshv-miR-K12-2 AACUGUAGUCCGGGUCGAUCUG 965 GACTACAGT 1902 ACTACAGT 2839CTACAGT 3776 kshv-miR-K12-3 UCACAUUCUGAGGACGGCAGCGA 966 CAGAATGTG 1903AGAATGTG 2840 GAATGTG 3777 kshv-miR-K12-3* UCGCGGUCACAGAAUGUGACA 967GTGACCGCG 1904 TGACCGCG 2841 GACCGCG 3778 kshv-miR-K12-4-3pUAGAAUACUGAGGCCUAGCUGA 968 CAGTATTCT 1905 AGTATTCT 2842 GTATTCT 3779kshv-miR-K12-4-5p AGCUAAACCGCAGUACUCUAGG 969 CGGTTTAGC 1906 GGTTTAGC2843 GTTTAGC 3780 kshv-miR-K12-5 UAGGAUGCCUGGAACUUGCCGG 970 AGGCATCCT1907 GGCATCCT 2844 GCATCCT 3781 kshv-miR-K12-6-3p UGAUGGUUUUCGGGCUGUUGAG971 AAAACCATC 1908 AAACCATC 2845 AACCATC 3782 kshv-miR-K12-6-5pCCAGCAGCACCUAAUCCAUCGG 972 GTGCTGCTG 1909 TGCTGCTG 2846 GCTGCTG 3783kshv-miR-K12-7 UGAUCCCAUGUUGCUGGCGCU 973 CATGGGATC 1910 ATGGGATC 2847TGGGATC 3784 kshv-miR-K12-8 UAGGCGCGACUGAGAGAGCACG 974 GTCGCGCCT 1911TCGCGCCT 2848 CGCGCCT 3785 kshv-miR-K12-9 CUGGGUAUACGCAGCUGCGUAA 975GTATACCCA 1912 TATACCCA 2849 ATACCCA 3786 kshv-miR-K12-9*ACCCAGCUGCGUAAACCCCGCU 976 GCAGCTGGG 1913 CAGCTGGG 2850 AGCTGGG 3787

The invention claimed is:
 1. A method for de-repression of a mRNA in acell, wherein the expression of the mRNA is repressed by a miRNA in saidcell, the method comprising administering an effective amount of anoligomer to said cell, to de-repress the expression of the mRNA, whereinthe oligomer has a length of 7, 8, 9 or 10 nucleotide units which arefully complementary to said miRNA, wherein each nucleotide unit is anLNA nucleotide unit, and wherein at least 75% of the internucleosidelinkages between the LNA nucleoside units are phosphorothioateinternucleoside linkages, and wherein the contiguous nucleotide sequenceof the oligomer is complementary to the seed sequence of said microRNA.2. The method according to claim 1, wherein all of the internucleosidelinkages between the LNA nucleoside units are phosphorothioateinternucleoside linkages.
 3. The method according to claim 1, whereinthe oligomer is 7 LNA nucleotides in length.
 4. The method according toclaim 1, wherein the oligomer is 8 LNA nucleotides in length.
 5. Themethod according to claim 1, wherein the oligomer is 9 LNA nucleotidesin length.
 6. The method according to claim 1, wherein the oligomer is10 LNA nucleotides in length.
 7. The method according to claim 2,wherein the oligomer is 7 LNA nucleotides in length.
 8. The methodaccording to claim 2, wherein the oligomer is 8 LNA nucleotides inlength.
 9. The method according to claim 2, wherein the oligomer is 9LNA nucleotides in length.
 10. The method according to claim 2, whereinthe oligomer is 10 LNA nucleotides in length.
 11. The method accordingto claim 1, wherein nucleotides 1-6 as measured from the 2′ end of theoligomer are fully complementary to the microRNA seed region.
 12. Themethod according to claim 1, wherein nucleotides 1-7 as measured fromthe 2′ end of the oligomer are fully complementary to the microRNA seedregion.
 13. The method according to claim 1, wherein nucleotides 2-7 asmeasured from the 2′ end of the oligomer are fully complementary to themicroRNA seed region.
 14. The method according to claim 2, whereinnucleotides 1-6 as measured from the 2′ end of the oligomer are fullycomplementary to the microRNA seed region.
 15. The method according toclaim 2, wherein nucleotides 1-7 as measured from the 2′ end of theoligomer are fully complementary to the microRNA seed region.
 16. Themethod according to claim 2, wherein nucleotides 2-7 as measured fromthe 2′ end of the oligomer are fully complementary to the microRNA seedregion.
 17. The method according to claim 1, wherein the microRNA is amammalian or viral microRNA, and the cell is a mammalian cell.
 18. Themethod according to claim 1, wherein the microRNA is a human microRNA,and the cell is a human cell.